Compositions and methods for inhibiting expression of huntingtin gene

ABSTRACT

The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the Huntingtin gene (HD gene), comprising an antisense strand having a nucleotide sequence which is less than 25 nucleotides in length and which is substantially complementary to at least a part of the HD gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases caused by the expression of the HD gene, or a mutant form thereof, using the pharmaceutical composition; and methods for inhibiting the expression of the huntingtin gene in a cell.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/731,555, filed Oct. 28, 2005, U.S. Provisional Application No.60/819,038, filed Jul. 7, 2006, and U.S. Provisional Application No.60/836,040, filed Aug. 7, 2006. The contents of each of these priorityapplications are incorporated herein by reference in their entirety.

SEQUENCE LISTING

This application incorporates by reference the sequence listing saved asan ASCII text file on CD-ROM. The sequence listing saved on CD-ROM wascreated on Oct. 27, 2006, and is identified as “14174-125001.txt.” Thefile contains 311 KB of data. Three identical copies of the sequencelisting have been submitted, including one “Computer-Readable Format”(CRF) and two “Official Copies” (Copy 1 and Copy 2).

FIELD OF THE INVENTION

This invention relates to double-stranded ribonucleic acid (dsRNA), andits use in mediating RNA interference to inhibit the expression of theHuntingtin gene.

BACKGROUND OF THE INVENTION

Recently, double-stranded RNA molecules (dsRNA) have been shown to blockgene expression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). WO 99/32619 (Fire et al.) discloses the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofgenes in C. elegans. dsRNA has also been shown to degrade target RNA inother organisms, including plants (see, e.g., WO 99/53050, Waterhouse etal.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D.,et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895,Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanismhas now become the focus for the development of a new class ofpharmaceutical agents for treating disorders that are caused by theaberrant regulation of genes or the expression of a mutant form of agene.

Huntington's disease is a progressive neurodegenerative disordercharacterized by motor disturbance, cognitive loss and psychiatricmanifestations (Martin and Gusella, N. Engl. J. Med. 315:1267-1276(1986). It is inherited in an autosomal dominant fashion, and affectsabout 1/10,000 individuals in most populations of European origin(Harper, P. S. et al., in Huntington's disease, W. B. Saunders,Philadelphia, 1991). The hallmark of Huntington's disease is adistinctive choreic movement disorder that typically has a subtle,insidious onset in the fourth to fifth decade of life and graduallyworsens over a course of 10 to 20 years until death. Occasionally,Huntington's disease is expressed in juveniles typically manifestingwith more severe symptoms including rigidity and a more rapid course.Juvenile onset of Huntington's disease is associated with apreponderance of paternal transmission of the disease allele. Theneuropathology of Huntington's disease also displays a distinctivepattern, with selective loss of neurons that is most severe in thecaudate and putamen regions of the brain. The biochemical basis forneuronal death in Huntington's disease has not yet been explained, andthere is consequently no treatment effective in delaying or preventingthe onset and progression of this devastating disorder.

Although an actual mechanism for Huntington's disease remains elusive,Huntington's disease has been shown to be an autosomal dominantneurodegenerative disorder caused by an expanding glutamine repeat in agene termed IT15 or Huntingtin (HD). Although this gene is widelyexpressed and is required for normal development, the pathology ofHuntington's disease is restricted to the brain, for reasons that remainpoorly understood. The Huntingtin gene product is expressed at similarlevels in patients and controls, and the genetics of the disordersuggest that the expansion of the polyglutamine repeat induces a toxicgain of function, perhaps through interactions with other cellularproteins.

Treatment for Huntington's disease is currently not available. Thechoreic movements and agitated behaviors may be suppressed, usually onlypartially, by antipsychotics (e.g., chlorpromazine 100 to 900 mg/day poor haloperidol 10 to 90 mg/day po) or reserpine begun with 0.1 mg/day poand increased until adverse effects of lethargy, hypotension, orparkinsonism occur.

Despite significant advances in the field of RNAi and Huntington'sdisease treatment, there remains a need for an agent that canselectively and efficiently silence the HD gene using the cell's ownRNAi machinery that has both high biological activity and in vivostability, and that can effectively inhibit expression of a targetHuntingtin gene.

SUMMARY OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of the HD genein a cell or mammal using such dsRNA. The invention also providescompositions and methods for treating diseases caused by the expressionof a mutant form of the HD gene. The dsRNA of the invention comprises anRNA strand (the antisense strand) having a region which is less than 30nucleotides in length and is substantially complementary to at leastpart of an mRNA transcript of the HD gene.

In one embodiment, the invention provides double-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of the HD gene. ThedsRNA comprises at least two sequences that are complementary to eachother. The dsRNA comprises a sense strand comprising a first sequenceand an antisense strand comprising a second sequence. The antisensestrand comprises a nucleotide sequence which is substantiallycomplementary to at least part of an mRNA encoding the huntingtinprotein, and the region of complementarity is less than 30 nucleotidesin length. The dsRNA, upon contacting with a cell expressing the HDgene, inhibits the expression of the HD gene by at least 20%.

For example, the dsRNA molecules of the invention can be comprised of afirst sequence of the dsRNA that is selected from the group consistingof the sense sequences of Tables 1, 2, 7, 8 or 10 and the secondsequence is selected from the group consisting of the antisensesequences of Tables 1, 2, 7, 8 or 10. The dsRNA molecules of theinvention can be comprised of naturally occurring nucleotides or can becomprised of at least one modified nucleotide, such as a 2′-O-methylmodified nucleotide, a nucleotide comprising a 5′-phosphorothioategroup, and a terminal nucleotide linked to a cholesteryl derivative ordodecanoic acid bisdecylamide group. Alternatively, the modifiednucleotide may be chosen from the group of: a 2′-deoxy-2′-fluoromodified nucleotide, a 2′-deoxy-modified nucleotide, a lockednucleotide, an abasic nucleotide, 2′-amino-modified nucleotide,2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate,and a non-natural base comprising nucleotide. Preferably, the firstsequence of said dsRNA is selected from the group consisting of thesense sequences of Table 2 and the second sequence is selected from thegroup consisting of the antisense sequences of Table 2.

In another embodiment, the invention provides a cell comprising one ofthe dsRNAs of the invention. The cell is preferably a mammalian cell,such as a human cell.

In another embodiment, the invention provides a pharmaceuticalcomposition for inhibiting the expression of the HD gene in an organism,comprising one or more of the dsRNA of the invention and apharmaceutically acceptable carrier.

In another embodiment, the invention provides a method for inhibitingthe expression of the HD gene in a cell, comprising the following steps:

-   -   (a) introducing into the cell a double-stranded ribonucleic acid        (dsRNA), wherein the dsRNA comprises at least two sequences that        are complementary to each other. The dsRNA comprises a sense        strand comprising a first sequence and an antisense strand        comprising a second sequence. The antisense strand comprises a        region of complementarity which is substantially complementary        to at least a part of a mRNA encoding the HD gene, and wherein        the region of complementarity is less than 30 nucleotides in        length and wherein the dsRNA, upon contact with a cell        expressing the HD gene, inhibits expression of the HD gene by at        least 20%; and    -   (b) maintaining the cell produced in step (a) for a time        sufficient to obtain degradation of the mRNA transcript of the        HD gene, thereby inhibiting expression of the HD gene in the        cell.

In another embodiment, the invention provides methods for treating,preventing or managing Huntington's disease comprising administering toa patient in need of such treatment, prevention or management atherapeutically or prophylactically effective amount of one or more ofthe dsRNAs of the invention.

In another embodiment, the invention provides vectors for inhibiting theexpression of the HD gene in a cell, comprising a regulatory sequenceoperably linked to a nucleotide sequence that encodes at least onestrand of one of the dsRNA of the invention.

In another embodiment, the invention provides cell comprising a vectorfor inhibiting the expression of the HD gene in a cell. The vectorcomprises a regulatory sequence operably linked to a nucleotide sequencethat encodes at least one strand of one of the dsRNA of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. In vitro activity of the dsRNAs provided in Table 2 againstendogenous human HD mRNA expression in HeLa cells.

FIG. 2. Activity of selected dsRNAs in reducing endogenous human HDprotein formation in HeLa cells.

FIG. 3. Stability of selected dsRNAs in cerebrospinal fluid (CSF) at 37°C.

FIG. 4. Long-term stability of dsRNAs AL-DP-5997, AL-DP-6000, AL-DP-6001and AL-DP-7100 in rat CSF

DETAILED DESCRIPTION OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of the HD genein a cell or mammal using the dsRNA. The invention also providescompositions and methods for treating diseases in a mammal caused by theexpression of the HD gene, or a mutant form thereof, using dsRNA. dsRNAdirects the sequence-specific degradation of mRNA through a processknown as RNA interference (RNAi). The process occurs in a wide varietyof organisms, including mammals and other vertebrates.

The dsRNA of the invention comprises an RNA strand (the antisensestrand) having a region which is less than 30 nucleotides in length andis substantially complementary to at least part of an mRNA transcript ofthe HD gene. The use of these dsRNAs enables the targeted degradation ofmRNAs of genes that are implicated in Huntington Disease. Usingcell-based and animal assays, the present inventors have demonstratedthat very low dosages of these dsRNA can specifically and efficientlymediate RNAi, resulting in significant inhibition of expression of theHD gene. Thus, the methods and compositions of the invention comprisingthese dsRNAs are useful for treating Huntington disease.

The following detailed description discloses how to make and use thedsRNA and compositions containing dsRNA to inhibit the expression of atarget HD gene, as well as compositions and methods for treatingdiseases and disorders caused by the expression of these genes. Thepharmaceutical compositions of the invention comprise a dsRNA having anantisense strand comprising a region of complementarity which is lessthan 30 nucleotides in length and is substantially complementary to atleast part of an RNA transcript of the HD gene, together with apharmaceutically acceptable carrier (Human HD mRNA (NM-002111), mouse HDmRNA (NM_(—)010414) and rat HD mRNA (U18650)).

Accordingly, certain aspects of the invention provide pharmaceuticalcompositions comprising the dsRNA of the invention together with apharmaceutically acceptable carrier, methods of using the compositionsto inhibit expression of the HD gene, and methods of using thepharmaceutical compositions to treat diseases caused by expression of amutant form of the HD gene.

I. Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.However, it will be understood that the term “ribonucleotide” or“nucleotide” can also refer to a modified nucleotide, as furtherdetailed below, or a surrogate replacement moiety. The skilled person iswell aware that guanine, cytosine, adenine, and uracil may be replacedby other moieties without substantially altering the base pairingproperties of an oligonucleotide comprising a nucleotide bearing suchreplacement moiety. For example, without limitation, a nucleotidecomprising inosine as its base may base pair with nucleotides containingadenine, cytosine, or uracil. Hence, nucleotides containing uracil,guanine, or adenine may be replaced in the nucleotide sequences of theinvention by a nucleotide containing, for example, inosine. Sequencescomprising such replacement moieties are embodiments of the invention.

The gene involved in Huntington's disease (IT-15) is located at the endof the short arm of chromosome 4. A mutation occurs in the coding regionof this gene and produces an unstable expanded trinucleotide repeat(cytosine-adenosine-guanosine), resulting in a protein with an expandedglutamate sequence. The normal and abnormal functions of this protein(termed huntingtin) are unknown. The abnormal huntingtin protein appearsto accumulate in neuronal nuclei of transgenic mice, but the causalrelationship of this accumulation to neuronal death is uncertain.

By “Huntingtin” or “HD” as used herein is meant, any Huntingtin protein,peptide, or polypeptide associated with the development or maintenanceof Huntington disease. The terms “Huntingtin” and “HD” also refer tonucleic acid sequences encoding any huntingtin protein, peptide, orpolypeptide, such as Huntingtin RNA or Huntingtin DNA (see for exampleVan Dellen et al., Jan. 24, 2004, Neurogenetics). For the Examples, theHD mRNA sequences used were Human HD mRNA (NM-002111), mouse HD mRNA(NM_(—)010414) and rat HD mRNA (U18650).

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof the HD gene, including mRNA that is a product of RNA processing of aprimary transcription product.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotidecomprising the first nucleotide sequence to the oligonucleotide orpolynucleotide comprising the second nucleotide sequence over the entirelength of the first and second nucleotide sequence. Such sequences canbe referred to as “fully complementary” with respect to each otherherein. However, where a first sequence is referred to as “substantiallycomplementary” with respect to a second sequence herein, the twosequences can be fully complementary, or they may form one or more, butpreferably not more than 4, 3 or 2 mismatched base pairs uponhybridization, while retaining the ability to hybridize under theconditions most relevant to their ultimate application. However, wheretwo oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs shall not be regarded asmismatches with regard to the determination of complementarity. Forexample, a dsRNA comprising one oligonucleotide 21 nucleotides in lengthand another oligonucleotide 23 nucleotides in length, wherein the longeroligonucleotide comprises a sequence of 21 nucleotides that is fullycomplementary to the shorter oligonucleotide, may yet be referred to as“fully complementary” for the purposes of the invention.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.

The terms “complementary”, “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a dsRNA and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide which is “substantially complementaryto at least part of” a messenger RNA (mRNA) refers to a polynucleotidewhich is substantially complementary to a contiguous portion of the mRNAof interest (e.g., encoding HD). For example, a polynucleotide iscomplementary to at least a part of a HD mRNA if the sequence issubstantially complementary to a non-interrupted portion of a mRNAencoding HD.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to aribonucleic acid molecule, or complex of ribonucleic acid molecules,having a duplex structure comprising two anti-parallel and substantiallycomplementary, as defined above, nucleic acid strands. The two strandsforming the duplex structure may be different portions of one larger RNAmolecule, or they may be separate RNA molecules. Where the two strandsare part of one larger molecule, and therefore are connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′end of the respective other strand forming the duplex structure,the connecting RNA chain is referred to as a “hairpin loop”. Where thetwo strands are connected covalently by means other than anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′end of the respective other strand forming the duplex structure,the connecting structure is referred to as a “linker”. The RNA strandsmay have the same or a different number of nucleotides. The maximumnumber of base pairs is the number of nucleotides in the shortest strandof the dsRNA. In addition to the duplex structure, a dsRNA may compriseone or more nucleotide overhangs.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches are most tolerated in the terminal regions and,if present, are preferably in a terminal region or regions, e.g., within6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

“Introducing into a cell”, when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell”, wherein the cell is part ofa living organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vitro introduction into a cell includes methods known in the art suchas electroporation and lipofection.

The terms “silence” and “inhibit the expression of”, in as far as theyrefer to the HD gene, herein refer to the at least partial suppressionof the expression of the HD gene, as manifested by a reduction of theamount of mRNA transcribed from the HD gene which may be isolated from afirst cell or group of cells in which the HD gene is transcribed andwhich has or have been treated such that the expression of the HD geneis inhibited, as compared to a second cell or group of cellssubstantially identical to the first cell or group of cells but whichhas or have not been so treated (control cells). The degree ofinhibition is usually expressed in terms of${\frac{\left( {{mRNA}\quad{in}\quad{control}\quad{cells}} \right) - \left( {{mRNA}\quad{in}\quad{treated}\quad{cells}} \right)}{\left( {{mRNA}\quad{in}\quad{control}\quad{cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to HD genetranscription, e.g. the amount of protein encoded by the HD gene whichis secreted by a cell, or the number of cells displaying a certainphenotype, e.g apoptosis. In principle, HD gene silencing may bedetermined in any cell expressing the target, either constitutively orby genomic engineering, and by any appropriate assay. However, when areference is needed in order to determine whether a given siRNA inhibitsthe expression of the HD gene by a certain degree and therefore isencompassed by the instant invention, the assay provided in the Examplesbelow shall serve as such reference.

For example, in certain instances, expression of the HD gene issuppressed by at least about 20%, 25%, 35%, or 50% by administration ofthe double-stranded oligonucleotide of the invention. In a preferredembodiment, the HD gene is suppressed by at least about 60%, 70%, or 80%by administration of the double-stranded oligonucleotide of theinvention. In a more preferred embodiment, the HD gene is suppressed byat least about 85%, 90%, or 95% by administration of the double-strandedoligonucleotide of the invention. In a most preferred embodiment, the HDgene is suppressed by at least about 98%, 99% or more by administrationof the double-stranded oligonucleotide of the invention.

As used herein, the term “treatment” refers to the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disorder, e.g., a disease or condition, asymptom of disease, or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve, or affect the disease, the symptoms of disease, or thepredisposition toward disease. A “patient” may be a human, but can alsobe a non-human animal. Treatment can refer to the reduction of any oneof the overt symptoms of Huntington's disease, such as dementia orpsychiatric disturbances, ranging from apathy and irritability tofull-blown bipolar or schizophreniform disorder, motor manifestationsinclude flicking movements of the extremities, a lilting gait, motorimpersistence (inability to sustain a motor act, such as tongueprotrusion), facial grimacing, ataxia, and dystonia.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management ofHuntington's disease or an overt symptom of the disease. The specificamount that is therapeutically effective can be readily determined byordinary medical practitioner, and may vary depending on factors knownin the art, such as, e.g. the type of Huntington's disease, thepatient's history and age, the stage of Huntington's disease, and theadministration of other anti-Huntington's disease agents.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

II. Double-Stranded Ribonucleic Acid (dsRNA)

In one embodiment, the invention provides double-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of the HD gene in acell or mammal, wherein the dsRNA comprises an antisense strandcomprising a region of complementarity which is complementary to atleast a part of an mRNA formed in the expression of the HD gene, andwherein the region of complementarity is less than 30 nucleotides inlength and wherein said dsRNA, upon contact with a cell expressing saidHD gene, inhibits the expression of said HD gene by at least 20%. ThedsRNA comprises two RNA strands that are sufficiently complementary tohybridize to form a duplex structure. One strand of the dsRNA (theantisense strand) comprises a region of complementarity that issubstantially complementary, and preferably fully complementary, to atarget sequence, derived from the sequence of an mRNA formed during theexpression of the HD gene, the other strand (the sense strand) comprisesa region which is complementary to the antisense strand, such that thetwo strands hybridize and form a duplex structure when combined undersuitable conditions. Preferably, the duplex structure is between 15 and30, more preferably between 18 and 25, yet more preferably between 19and 24, and most preferably between 21 and 23 base pairs in length.Similarly, the region of complementarity to the target sequence isbetween 15 and 30, more preferably between 18 and 25, yet morepreferably between 19 and 24, and most preferably between 21 and 23nucleotides in length. The dsRNA of the invention may further compriseone or more single-stranded nucleotide overhang(s). The dsRNA can besynthesized by standard methods known in the art as further discussedbelow, e.g., by use of an automated DNA synthesizer, such as arecommercially available from, for example, Biosearch, Applied Biosystems,Inc. In a preferred embodiment, the HD gene is the human HD gene. Inspecific embodiments, the antisense strand of the dsRNA comprises theantisense sequences of Tables 1, 2, 7, 8 or 10 and the second sequenceis selected from the group consisting of the sense sequences of Tables1, 2, 7, 8 or 10.

In further embodiments, the dsRNA comprises at least one nucleotidesequence selected from the groups of sequences provided in Tables 1, 2,7, 8 or 10. In other embodiments, the dsRNA comprises at least twosequences selected from this group, wherein one of the at least twosequences is complementary to another of the at least two sequences, andone of the at least two sequences is substantially complementary to asequence of an mRNA generated in the expression of the HD gene.Preferably, the dsRNA comprises two oligonucleotides, wherein oneoligonucleotide is described by Tables 1, 2, 7, 8 or 10 and the secondoligonucleotide is described Tables 1, 2, 7, 8 or 10.

The skilled person is well aware that dsRNAs comprising a duplexstructure of between 20 and 23, but specifically 21, base pairs havebeen hailed as particularly effective in inducing RNA interference(Elbashir et al., EMBO 2001, 20:6877-6888). However, others have foundthat shorter or longer dsRNAs can be effective as well. In theembodiments described above, by virtue of the nature of theoligonucleotide sequences provided in Tables 1, 2, 7, 8 or 10, thedsRNAs of the invention can comprise at least one strand of a length ofminimally 21 nt. It can be reasonably expected that shorter dsRNAscomprising one of the sequences of Tables 1, 2, 7, 8 or 10 minus only afew nucleotides on one or both ends may be similarly effective ascompared to the dsRNAs described above. Hence, dsRNAs comprising apartial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguousnucleotides from one of the sequences of Tables 1, 2, 7, 8 or 10, anddiffering in their ability to inhibit the expression of the HD gene in aFACS assay as described herein below by not more than 5, 10, 15, 20, 25,or 30% inhibition from a dsRNA comprising the full sequence, arecontemplated by the invention.

The dsRNA of the invention can contain one or more mismatches to thetarget sequence. In a preferred embodiment, the dsRNA of the inventioncontains no more than 3 mismatches. If the antisense strand of the dsRNAcontains mismatches to a target sequence, it is preferable that the areaof mismatch not be located in the center of the region ofcomplementarity. If the antisense strand of the dsRNA containsmismatches to the target sequence, it is preferable that the mismatch berestricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or1 nucleotide from either the 5′ or 3+ end of the region ofcomplementarity. For example, for a 23 nucleotide dsRNA strand which iscomplementary to a region of the HD gene, the dsRNA preferably does notcontain any mismatch within the central 13 nucleotides. The methodsdescribed within the invention can be used to determine whether a dsRNAcontaining a mismatch to a target sequence is effective in inhibitingthe expression of the HD gene. Consideration of the efficacy of dsRNAswith mismatches in inhibiting expression of the HD gene is important,especially if the particular region of complementarity in the HD gene isknown to have polymorphic sequence variation within the population.

In one embodiment, at least one end of the dsRNA has a single-strandednucleotide overhang of 1 to 4, preferably 1 or 2 nucleotides. dsRNAshaving at least one nucleotide overhang have unexpectedly superiorinhibitory properties than their blunt-ended counterparts. Moreover, thepresent inventors have discovered that the presence of only onenucleotide overhang strengthens the interference activity of the dsRNA,without affecting its overall stability. dsRNA having only one overhanghas proven particularly stable and effective in vivo, as well as in avariety of cells, cell culture mediums, blood, and serum. Preferably,the single-stranded overhang is located at the 3′-terminal end of theantisense strand or, alternatively, at the 3′-terminal end of the sensestrand. The dsRNA may also have a blunt end, preferably located at the5′-end of the antisense strand. Such dsRNAs have improved stability andinhibitory activity, thus allowing administration at low dosages, i.e.,less than 5 mg/kg body weight of the recipient per day. Preferably, theantisense strand of the dsRNA has a nucleotide overhang at the 3′-end,and the 5′-end is blunt. In another embodiment, one or more of thenucleotides in the overhang is replaced with a nucleoside thiophosphate.

In yet another embodiment, the dsRNA is chemically modified to enhancestability. The nucleic acids of the invention may be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Chemical modifications may include,but are not limited to 2′ modifications, introduction of non-naturalbases, covalent attachment to a ligand, and replacement of phosphatelinkages with thiophosphate linkages. In this embodiment, the integrityof the duplex structure is strengthened by at least one, and preferablytwo, chemical linkages. Chemical linking may be achieved by any of avariety of well-known techniques, for example by introducing covalent,ionic or hydrogen bonds; hydrophobic interactions, van der Waals orstacking interactions; by means of metal-ion coordination, or throughuse of purine analogues. Preferably, the chemical groups that can beused to modify the dsRNA include, without limitation, methylene blue;bifunctional groups, preferably bis-(2-chloroethyl)amine;N-acetyl-N′-(p-glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen. Inone preferred embodiment, the linker is a hexa-ethylene glycol linker.In this case, the dsRNA are produced by solid phase synthesis and thehexa-ethylene glycol linker is incorporated according to standardmethods (e.g., Williams, D. J., and K. B. Hall, Biochem. (1996)35:14665-14670). In a particular embodiment, the 5′-end of the antisensestrand and the 3′-end of the sense strand are chemically linked via ahexaethylene glycol linker. In another embodiment, at least onenucleotide of the dsRNA comprises a phosphorothioate orphosphorodithioate groups. The chemical bond at the ends of the dsRNA ispreferably formed by triple-helix bonds. Table 2 provides examples ofmodified RNAi agents of the invention.

In certain embodiments, a chemical bond may be formed by means of one orseveral bonding groups, wherein such bonding groups are preferablypoly-(oxyphosphinicooxy-1,3-propandiol)- and/or polyethylene glycolchains. In other embodiments, a chemical bond may also be formed bymeans of purine analogs introduced into the double-stranded structureinstead of purines. In further embodiments, a chemical bond may beformed by azabenzene units introduced into the double-strandedstructure. In still further embodiments, a chemical bond may be formedby branched nucleotide analogs instead of nucleotides introduced intothe double-stranded structure. In certain embodiments, a chemical bondmay be induced by ultraviolet light.

In yet another embodiment, the nucleotides at one or both of the twosingle strands may be modified to prevent or inhibit the activation ofcellular enzymes, such as, for example, without limitation, certainnucleases. Techniques for inhibiting the activation of cellular enzymesare known in the art including, but not limited to, 2′-aminomodifications, 2′-amino sugar modifications, 2′-F sugar modifications,2′-F modifications, 2′-alkyl sugar modifications, uncharged backbonemodifications, morpholino modifications, 2′-O-methyl modifications, andphosphoramidate (see, e.g., Wagner, Nat. Med. (1995) 1:1116-8). Thus, atleast one 2′-hydroxyl group of the nucleotides on a dsRNA is replaced bya chemical group, preferably by a 2′-amino or a 2′-methyl group. Also,at least one nucleotide may be modified to form a locked nucleotide.Such locked nucleotide contains a methylene bridge that connects the2′-oxygen of ribose with the 4′-carbon of ribose. Oligonucleotidescontaining the locked nucleotide are described in (Koshkin, A. A., etal., Tetrahedron (1998), 54: 3607-3630 and Obika, S. et al., TetrahedronLett. (1998), 39: 5401-5404). Introduction of a locked nucleotide intoan oligonucleotide improves the affinity for complementary sequences andincreases the melting temperature by several degrees (Braasch, D. A. andD. R. Corey, Chem. Biol. (2001), 8:1-7).

Conjugating a ligand to a dsRNA can enhance its cellular absorption. Incertain instances, a hydrophobic ligand is conjugated to the dsRNA tofacilitate direct permeation of the cellular membrane. Alternatively,the ligand conjugated to the dsRNA is a substrate for receptor-mediatedendocytosis. These approaches have been used to facilitate cellpermeation of antisense oligonucleotides. For example, cholesterol hasbeen conjugated to various antisense oligonucleotides resulting incompounds that are substantially more active compared to theirnon-conjugated analogs. See M. Manoharan Antisense & Nucleic Acid DrugDevelopment 2002, 12, 103. Other lipophilic compounds that have beenconjugated to oligonucleotides include 1-pyrene butyric acid,1,3-bis-O-(hexadecyl)glycerol, and menthol. One example of a ligand forreceptor-mediated endocytosis is folic acid. Folic acid enters the cellby folate-receptor-mediated endocytosis. dsRNA compounds bearing folicacid would be efficiently transported into the cell via thefolate-receptor-mediated endocytosis. Li and coworkers report thatattachment of folic acid to the 3′-terminus of an oligonucleotideresulted in an 8-fold increase in cellular uptake of theoligonucleotide. Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res. 1998,15, 1540. Other ligands that have been conjugated to oligonucleotidesinclude polyethylene glycols, carbohydrate clusters, cross-linkingagents, porphyrin conjugates, and delivery peptides.

In certain instances, conjugation of a cationic ligand tooligonucleotides often results in improved resistance to nucleases.Representative examples of cationic ligands are propylammonium anddimethylpropylammonium. Interestingly, antisense oligonucleotides werereported to retain their high binding affinity to mRNA when the cationicligand was dispersed throughout the oligonucleotide. See M. ManoharanAntisense & Nucleic Acid Drug Development 2002, 12, 103 and referencestherein.

The ligand-conjugated dsRNA of the invention may be synthesized by theuse of a dsRNA that bears a pendant reactive functionality, such as thatderived from the attachment of a linking molecule onto the dsRNA. Thisreactive oligonucleotide may be reacted directly withcommercially-available ligands, ligands that are synthesized bearing anyof a variety of protecting groups, or ligands that have a linking moietyattached thereto. The methods of the invention facilitate the synthesisof ligand-conjugated dsRNA by the use of, in some preferred embodiments,nucleoside monomers that have been appropriately conjugated with ligandsand that may further be attached to a solid-support material. Suchligand-nucleoside conjugates, optionally attached to a solid-supportmaterial, are prepared according to some preferred embodiments of themethods of the invention via reaction of a selected serum-binding ligandwith a linking moiety located on the 5′ position of a nucleoside oroligonucleotide. In certain instances, an dsRNA bearing an aralkylligand attached to the 3′-terminus of the dsRNA is prepared by firstcovalently attaching a monomer building block to a controlled-pore-glasssupport via a long-chain aminoalkyl group. Then, nucleotides are bondedvia standard solid-phase synthesis techniques to the monomerbuilding-block bound to the solid support. The monomer building blockmay be a nucleoside or other organic compound that is compatible withsolid-phase synthesis.

The dsRNA used in the conjugates of the invention may be convenientlyand routinely made through the well-known technique of solid-phasesynthesis. Equipment for such synthesis is sold by several vendorsincluding, for example, Applied Biosystems (Foster City, Calif.). Anyother means for such synthesis known in the art may additionally oralternatively be employed. It is also known to use similar techniques toprepare other oligonucleotides, such as the phosphorothioates andalkylated derivatives.

Teachings regarding the synthesis of particular modifiedoligonucleotides may be found in the following U.S. Pat. Nos. 5,138,045and 5,218,105, drawn to polyamine conjugated oligonucleotides; U.S. Pat.No. 5,212,295, drawn to monomers for the preparation of oligonucleotideshaving chiral phosphorus linkages; U.S. Pat. Nos. 5,378,825 and5,541,307, drawn to oligonucleotides having modified backbones; U.S.Pat. No. 5,386,023, drawn to backbone-modified oligonucleotides and thepreparation thereof through reductive coupling; U.S. Pat. No. 5,457,191,drawn to modified nucleobases based on the 3-deazapurine ring system andmethods of synthesis thereof; U.S. Pat. No. 5,459,255, drawn to modifiednucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302,drawn to processes for preparing oligonucleotides having chiralphosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleicacids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides havingβ-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods andmaterials for the synthesis of oligonucleotides; U.S. Pat. No.5,578,718, drawn to nucleosides having alkylthio groups, wherein suchgroups may be used as linkers to other moieties attached at any of avariety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and5,599,797, drawn to oligonucleotides having phosphorothioate linkages ofhigh chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for thepreparation of 2′-O-alkyl guanosine and related compounds, including2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn tooligonucleotides having N-2 substituted purines; U.S. Pat. No.5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat.No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to conjugated4′-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240, and5,610,289, drawn to backbone-modified oligonucleotide analogs; U.S. Pat.Nos. 6,262,241, and 5,459,255, drawn to, inter alia, methods ofsynthesizing 2′-fluoro-oligonucleotides.

In the ligand-conjugated dsRNA and ligand-molecule bearingsequence-specific linked nucleosides of the invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-buildingblocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide.Oligonucleotide conjugates bearing a variety of molecules such assteroids, vitamins, lipids and reporter molecules, has previously beendescribed (see Manoharan et al., PCT Application WO 93/07883). In apreferred embodiment, the oligonucleotides or linked nucleosides of theinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

The incorporation of a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-allyl,2′-O-aminoalkyl or 2′-deoxy-2′-fluoro group in nucleosides of anoligonucleotide confers enhanced hybridization properties to theoligonucleotide. Further, oligonucleotides containing phosphorothioatebackbones have enhanced nuclease stability. Thus, functionalized, linkednucleosides of the invention can be augmented to include either or botha phosphorothioate backbone or a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl,2′-O-aminoalkyl, 2′-O-allyl or 2′-deoxy-2′-fluoro group.

In some preferred embodiments, functionalized nucleoside sequences ofthe invention possessing an amino group at the 5′-terminus are preparedusing a DNA synthesizer, and then reacted with an active esterderivative of a selected ligand. Active ester derivatives are well knownto those skilled in the art. Representative active esters includeN-hydrosuccinimide esters, tetrafluorophenolic esters,pentafluorophenolic esters and pentachlorophenolic esters. The reactionof the amino group and the active ester produces an oligonucleotide inwhich the selected ligand is attached to the 5′-position through alinking group. The amino group at the 5′-terminus can be preparedutilizing a 5′-Amino-Modifier C6 reagent. In a preferred embodiment,ligand molecules may be conjugated to oligonucleotides at the5′-position by the use of a ligand-nucleoside phosphoramidite whereinthe ligand is linked to the 5′-hydroxy group directly or indirectly viaa linker. Such ligand-nucleoside phosphoramidites are typically used atthe end of an automated synthesis procedure to provide aligand-conjugated oligonucleotide bearing the ligand at the 5′-terminus.

In one preferred embodiment of the methods of the invention, thepreparation of ligand conjugated oligonucleotides commences with theselection of appropriate precursor molecules upon which to construct theligand molecule. Typically, the precursor is an appropriately-protectedderivative of the commonly-used nucleosides. For example, the syntheticprecursors for the synthesis of the ligand-conjugated oligonucleotidesof the invention include, but are not limited to,2′-aminoalkoxy-5′-ODMT-nucleosides,2′-6-aminoalkylamino-5′-ODMT-nucleosides,5′-6-aminoalkoxy-2′-deoxy-nucleosides,5′-6-aminoalkoxy-2-protected-nucleosides,3′-6-aminoalkoxy-5′-ODMT-nucleosides, and3′-aminoalkylamino-5′-ODMT-nucleosides that may be protected in thenucleobase portion of the molecule. Methods for the synthesis of suchamino-linked protected nucleoside precursors are known to those ofordinary skill in the art.

In many cases, protecting groups are used during the preparation of thecompounds of the invention. As used herein, the term “protected” meansthat the indicated moiety has a protecting group appended thereon. Insome preferred embodiments of the invention, compounds contain one ormore protecting groups. A wide variety of protecting groups can beemployed in the methods of the invention. In general, protecting groupsrender chemical functionalities inert to specific reaction conditions,and can be appended to and removed from such functionalities in amolecule without substantially damaging the remainder of the molecule.

Representative hydroxyl protecting groups, for example, are disclosed byBeaucage et al. (Tetrahedron, 1992, 48:2223-2311). Further hydroxylprotecting groups, as well as other representative protecting groups,are disclosed in Greene and Wuts, Protective Groups in OrganicSynthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991, andOligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed.,IRL Press, N.Y., 1991.

Examples of hydroxyl protecting groups include, but are not limited to,t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl,p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl,diphenylmethyl, p,p′-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl,trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetate,chloroacetate, trichloroacetate, trifluoroacetate, pivaloate, benzoate,p-phenylbenzoate, 9-fluorenylmethyl carbonate, mesylate and tosylate.

Amino-protecting groups stable to acid treatment are selectively removedwith base treatment, and are used to make reactive amino groupsselectively available for substitution. Examples of such groups are theFmoc (E. Atherton and R. C. Sheppard in The Peptides, S. Udenfriend, J.Meienhofer, Eds., Academic Press, Orlando, 1987, volume 9, p. 1) andvarious substituted sulfonylethyl carbamates exemplified by the Nscgroup (Samukov et al., Tetrahedron Lett., 1994, 35:7821; Verhart andTesser, Rec. Trav. Chim. Pays-Bas, 1987, 107:621).

Additional amino-protecting groups include, but are not limited to,carbamate protecting groups, such as 2-trimethylsilylethoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc),and benzyloxycarbonyl (Cbz); amide protecting groups, such as formyl,acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamideprotecting groups, such as 2-nitrobenzenesulfonyl; and imine and cyclicimide protecting groups, such as phthalimido and dithiasuccinoyl.Equivalents of these amino-protecting groups are also encompassed by thecompounds and methods of the invention.

Many solid supports are commercially available and one of ordinary skillin the art can readily select a solid support to be used in thesolid-phase synthesis steps. In certain embodiments, a universal supportis used. A universal support allows for preparation of oligonucleotideshaving unusual or modified nucleotides located at the 3′-terminus of theoligonucleotide. Universal Support 500 and Universal Support II areuniversal supports that are commercially available from Glen Research,22825 Davis Drive, Sterling, Va. For further details about universalsupports see Scott et al., Innovations and Perspectives in solid-phaseSynthesis, 3rd International Symposium, 1994, Ed. Roger Epton, MayflowerWorldwide, 115-124]; Azhayev, A. V. Tetrahedron 1999, 55, 787-800; andAzhayev and Antopolsky Tetrahedron 2001, 57, 4977-4986. In addition, ithas been reported that the oligonucleotide can be cleaved from theuniversal support under milder reaction conditions when oligonucleotideis bonded to the solid support via a syn-1,2-acetoxyphosphate groupwhich more readily undergoes basic hydrolysis. See Guzaev, A. I.;Manoharan, M. J. Am. Chem. Soc. 2003, 125, 2380.

The nucleosides are linked by phosphorus-containing ornon-phosphorus-containing covalent internucleoside linkages. For thepurposes of identification, such conjugated nucleosides can becharacterized as ligand-bearing nucleosides or ligand-nucleosideconjugates. The linked nucleosides having an aralkyl ligand conjugatedto a nucleoside within their sequence will demonstrate enhanced dsRNAactivity when compared to like dsRNA compounds that are not conjugated.

The aralkyl-ligand-conjugated oligonucleotides of the invention alsoinclude conjugates of oligonucleotides and linked nucleosides whereinthe ligand is attached directly to the nucleoside or nucleotide withoutthe intermediacy of a linker group. The ligand may preferably beattached, via linking groups, at a carboxyl, amino or oxo group of theligand. Typical linking groups may be ester, amide or carbamate groups.

Specific examples of preferred modified oligonucleotides envisioned foruse in the ligand-conjugated oligonucleotides of the invention includeoligonucleotides containing modified backbones or non-naturalinternucleoside linkages. As defined here, oligonucleotides havingmodified backbones or internucleoside linkages include those that retaina phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of the invention,modified oligonucleotides that do not have a phosphorus atom in theirintersugar backbone can also be considered to be oligonucleosides.

Specific oligonucleotide chemical modifications are described below. Itis not necessary for all positions in a given compound to be uniformlymodified. Conversely, more than one modifications may be incorporated ina single dsRNA compound or even in a single nucleotide thereof.

Preferred modified internucleoside linkages or backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free-acidforms are also included.

Representative United States patents relating to the preparation of theabove phosphorus-atom-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,625,050; and 5,697,248, each of which is hereinincorporated by reference.

Preferred modified internucleoside linkages or backbones that do notinclude a phosphorus atom therein (i.e., oligonucleosides) havebackbones that are formed by short chain alkyl or cycloalkyl intersugarlinkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages,or one or more short chain heteroatomic or heterocyclic intersugarlinkages. These include those having morpholino linkages (formed in partfrom the sugar portion of a nucleoside); siloxane backbones; sulfide,sulfoxide and sulfone backbones; formacetyl and thioformacetylbackbones; methylene formacetyl and thioformacetyl backbones; alkenecontaining backbones; sulfamate backbones; methyleneimino andmethylenehydrazino backbones; sulfonate and sulfonamide backbones; amidebackbones; and others having mixed N, O, S and CH₂ component parts.

Representative United States patents relating to the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleoside units arereplaced with novel groups. The nucleobase units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigonucleotide, an oligonucleotide mimetic, that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotideis replaced with an amide-containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to atoms of the amide portion of the backbone.Representative United States patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Further teaching of PNA compounds can be found in Nielsen etal., Science, 1991, 254, 1497.

Some preferred embodiments of the invention employ oligonucleotides withphosphorothioate linkages and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂—, and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

The oligonucleotides employed in the ligand-conjugated oligonucleotidesof the invention may additionally or alternatively comprise nucleobase(often referred to in the art simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C), and uracil (U). Modified nucleobasesinclude other synthetic and natural nucleobases, such as5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in the Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligonucleotides of the invention. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-Methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Id., pages 276-278) and are presentlypreferred base substitutions, even more particularly when combined with2′-methoxyethyl sugar modifications.

Representative United States patents relating to the preparation ofcertain of the above-noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,681,941; and 5,808,027; all of which are herebyincorporated by reference.

In certain embodiments, the oligonucleotides employed in theligand-conjugated oligonucleotides of the invention may additionally oralternatively comprise one or more substituted sugar moieties. Preferredoligonucleotides comprise one of the following at the 2′ position: OH;F; O—, S—, or N-alkyl, O—, S—, or N-alkenyl, or O, S— or N-alkynyl,wherein the alkyl, alkenyl and alkynyl may be substituted orunsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl.Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃,O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. apreferred modification includes 2′-methoxyethoxy [2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE] (Martin et al., Helv. Chim.Acta, 1995, 78, 486), i.e., an alkoxyalkoxy group. A further preferredmodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, as described in U.S. Pat. No. 6,127,533,filed on Jan. 30, 1998, the contents of which are incorporated byreference.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides.

As used herein, the term “sugar substituent group” or “2′-substituentgroup” includes groups attached to the 2′-position of the ribofuranosylmoiety with or without an oxygen atom. Sugar substituent groups include,but are not limited to, fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy,protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole andpolyethers of the formula (O-alkyl)_(m), wherein m is 1 to about 10.Preferred among these polyethers are linear and cyclic polyethyleneglycols (PEGs), and (PEG)-containing groups, such as crown ethers andthose which are disclosed by Ouchi et al. (Drug Design and Discovery1992, 9:93); Ravasio et al. (J. Org. Chem. 1991, 56:4329); and Delgardoet. al. (Critical Reviews in Therapeutic Drug Carrier Systems 1992,9:249), each of which is hereby incorporated by reference in itsentirety. Further sugar modifications are disclosed by Cook(Anti-Huntingtin disease Drug Design, 1991, 6:585-607). Fluoro, O-alkyl,O-alkylamino, O-alkyl imidazole, O-alkylaminoalkyl, and alkyl aminosubstitution is described in U.S. Pat. No. 6,166,197, entitled“Oligomeric Compounds having Pyrimidine Nucleotide(s) with 2′ and 5′Substitutions,” hereby incorporated by reference in its entirety.

Additional sugar substituent groups amenable to the invention include2′-SR and 2′-NR₂ groups, wherein each R is, independently, hydrogen, aprotecting group or substituted or unsubstituted alkyl, alkenyl, oralkynyl. 2′-SR Nucleosides are disclosed in U.S. Pat. No. 5,670,633,hereby incorporated by reference in its entirety. The incorporation of2′-SR monomer synthons is disclosed by Hamm et al. (J. Org. Chem., 1997,62:3415-3420). 2′-NR nucleosides are disclosed by Goettingen, M., J.Org. Chem., 1996, 61, 6273-6281; and Polushin et al., Tetrahedron Lett.,1996, 37, 3227-3230. Further representative 2′-substituent groupsamenable to the invention include those having one of formula I or II:

wherein,

E is C₁-C₁₀ alkyl, N(Q₃)(Q₄) or N═C (Q₃)(Q₄); each Q₃ and Q₄ is,independently, H, C₁-C₁₀ alkyl, dialkylaminoalkyl, a nitrogen protectinggroup, a tethered or untethered conjugate group, a linker to a solidsupport; or Q₃ and Q₄, together, form a nitrogen protecting group or aring structure optionally including at least one additional heteroatomselected from N and O;

q₁ is an integer from 1 to 10;

q₂ is an integer from 1 to 10;

q₃is 0 or 1;

q₄ is 0, 1 or 2;

each Z₁, Z₂ and Z₃ is, independently, C₄-C₇ cycloalkyl, C₅-C₁₄ aryl orC₃-C₁₅ heterocyclyl, wherein the heteroatom in said heterocyclyl groupis selected from oxygen, nitrogen and sulfur;

Z₄ is OM₁, SM₁, or N(M₁)₂; each M₁ is, independently, H, C₁-C₈ alkyl,C₁-C₈ haloalkyl, C(═NH)N(H)M₂, C(═O)N(H)M₂ or OC(═O)N(H)M₂; M₂ is H orC₁-C₈ alkyl; and

Z₅ is C₁-C₁₀ alkyl, C₁-C₁₀ haloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,C₆-C₁₄ aryl, N(Q₃)(Q₄), OQ₃, halo, SQ₃ or CN.

Representative 2′-O-sugar substituent groups of formula I are disclosedin U.S. Pat. No. 6,172,209, entitled “Capped 2′-OxyethoxyOligonucleotides,” hereby incorporated by reference in its entirety.Representative cyclic 2′-O-sugar substituent groups of formula II aredisclosed in U.S. Pat. No. 6,271,358, entitled “RNA Targeted 2′-ModifiedOligonucleotides that are Conformationally Preorganized,” herebyincorporated by reference in its entirety.

Sugars having O-substitutions on the ribosyl ring are also amenable tothe invention. Representative substitutions for ring O include, but arenot limited to, S, CH₂, CHF, and CF₂. See, e.g., Secrist et al.,Abstract 21, Program & Abstracts, Tenth International Roundtable,Nucleosides, Nucleotides and their Biological Applications, Park City,Utah, Sep. 16-20, 1992.

Oligonucleotides may also have sugar mimetics, such as cyclobutylmoieties, in place of the pentofuranosyl sugar. Representative UnitedStates patents relating to the preparation of such modified sugarsinclude, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800;5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,700,920; and5,859,221, all of which are hereby incorporated by reference.

Additional modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide. For example, one additional modification of theligand-conjugated oligonucleotides of the invention involves chemicallylinking to the oligonucleotide one or more additional non-ligandmoieties or conjugates which enhance the activity, cellular distributionor cellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties, such as a cholesterol moiety (Letsingeret al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid(Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993,3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992,20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBSLett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49), aphospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923).

Representative United States patents relating to the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941, each of whichis herein incorporated by reference.

The invention also includes compositions employing oligonucleotides thatare substantially chirally pure with regard to particular positionswithin the oligonucleotides. Examples of substantially chirally pureoligonucleotides include, but are not limited to, those havingphosphorothioate linkages that are at least 75% Sp or Rp (Cook et al.,U.S. Pat. No. 5,587,361) and those having substantially chirally pure(Sp or Rp) alkylphosphonate, phosphoramidate or phosphotriester linkages(Cook, U.S. Pat. Nos. 5,212,295 and 5,521,302).

In certain instances, the oligonucleotide may be modified by anon-ligand group. A number of non-ligand molecules have been conjugatedto oligonucleotides in order to enhance the activity, cellulardistribution or cellular uptake of the oligonucleotide, and proceduresfor performing such conjugations are available in the scientificliterature. Such non-ligand moieties have included lipid moieties, suchas cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989,86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994,4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann.N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem.Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov etal., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993,75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923). Representative United States patents thatteach the preparation of such oligonucleotide conjugates have beenlisted above. Typical conjugation protocols involve the synthesis ofoligonucleotides bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction may be performed either with the oligonucleotidestill bound to the solid support or following cleavage of theoligonucleotide in solution phase. Purification of the oligonucleotideconjugate by HPLC typically affords the pure conjugate.

Alternatively, the molecule being conjugated may be converted into abuilding block, such as a phosphoramidite, via an alcohol group presentin the molecule or by attachment of a linker bearing an alcohol groupthat may be phosphitylated.

Importantly, each of these approaches may be used for the synthesis ofligand conjugated oligonucleotides. Aminolinked oligonucleotides may becoupled directly with ligand via the use of coupling reagents orfollowing activation of the ligand as an NHS or pentfluorophenolateester. Ligand phosphoramidites may be synthesized via the attachment ofan aminohexanol linker to one of the carboxyl groups followed byphosphitylation of the terminal alcohol functionality. Other linkers,such as cysteamine, may also be utilized for conjugation to achloroacetyl linker present on a synthesized oligonucleotide.

III. Pharmaceutical Compositions Comprising dsRNA

In one embodiment, the invention provides pharmaceutical compositionscomprising a dsRNA, as described in the preceding section, and apharmaceutically acceptable carrier, as described below. Thepharmaceutical composition comprising the dsRNA is useful for treating adisease or disorder associated with the expression or activity of the HDgene.

In another embodiment, the invention provides pharmaceuticalcompositions comprising at least two dsRNAs, designed to targetdifferent regions of the HD gene, and a pharmaceutically acceptablecarrier. In this embodiment, the individual dsRNAs are prepared asdescribed in the preceding section, which is incorporated by referenceherein. One dsRNA can have a nucleotide sequence which is substantiallycomplementary to at least one part of the HD gene; additional dsRNAs areprepared, each of which has a nucleotide sequence that is substantiallycomplementary to different part of the HD gene. The multiple dsRNAs maybe combined in the same pharmaceutical composition, or formulatedseparately. If formulated individually, the compositions containing theseparate dsRNAs may comprise the same or different carriers, and may beadministered using the same or different routes of administration.Moreover, the pharmaceutical compositions comprising the individualdsRNAs may be administered substantially simultaneously, sequentially,or at preset intervals throughout the day or treatment period.

The pharmaceutical compositions of the invention are administered indosages sufficient to inhibit expression of the HD gene. The presentinventors have found that, because of their improved efficiency,compositions comprising the dsRNA of the invention can be administeredat surprisingly low dosages. A maximum dosage of 5 mg dsRNA per kilogrambody weight of recipient per day is sufficient to inhibit or completelysuppress expression of the HD gene.

In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0milligrams per kilogram body weight of the recipient per day, preferablyin the range of 0.1 to 200 micrograms per kilogram body weight per day,more preferably in the range of 0.1 to 100 micrograms per kilogram bodyweight per day, even more preferably in the range of 1.0 to 50micrograms per kilogram body weight per day, and most preferably in therange of 1.0 to 25 micrograms per kilogram body weight per day. Thepharmaceutical composition may be administered once daily, or the dsRNAmay be administered as two, three, four, five, six or more sub-doses atappropriate intervals throughout the day. In that case, the dsRNAcontained in each sub-dose must be correspondingly smaller in order toachieve the total daily dosage. The dosage unit can also be compoundedfor delivery over several days, e.g., using a conventional sustainedrelease formulation which provides sustained release of the dsRNA over aseveral day period. Sustained release formulations are well known in theart. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as Huntington's disease. Suchmodels are used for in vivo testing of dsRNA, as well as for determininga therapeutically effective dose.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intracranial (includingintraparenchymal and intraventricular), intrathecal, epidural,intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal,airway (aerosol), nasal, rectal, vaginal and topical (including buccaland sublingual) administration. In preferred embodiments, thepharmaceutical compositions are administered by intravenous, intrathecalor intracranial infusion or injection.

For intramuscular, intracranial, intrathecal, subcutaneous andintravenous use, the pharmaceutical compositions of the invention willgenerally be provided in sterile aqueous solutions or suspensions,buffered to an appropriate pH and isotonicity. Suitable aqueous vehiclesinclude Ringer's solution and isotonic sodium chloride. In a preferredembodiment, the carrier consists exclusively of an aqueous buffer. Inthis context, “exclusively” means no auxiliary agents or encapsulatingsubstances are present which might affect or mediate uptake of dsRNA inthe cells that express the HD gene. Such substances include, forexample, micellar structures, such as liposomes or capsids, as describedbelow. Surprisingly, the present inventors have discovered thatcompositions containing only naked dsRNA and a physiologicallyacceptable solvent are taken up by cells, where the dsRNA effectivelyinhibits expression of the HD gene. Although microinjection,lipofection, viruses, viroids, capsids, capsoids, or other auxiliaryagents are required to introduce dsRNA into cell cultures, surprisinglythese methods and agents are not necessary for uptake of dsRNA in vivo.Aqueous suspensions according to the invention may include suspendingagents such as cellulose derivatives, sodium alginate,polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such aslecithin. Suitable preservatives for aqueous suspensions include ethyland n-propyl p-hydroxybenzoate.

The pharmaceutical compositions useful according to the invention alsoinclude encapsulated formulations to protect the dsRNA against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811; PCT publication WO91/06309; and European patent publication EP-A-43075, which areincorporated by reference herein.

Using the small interfering RNA vectors previously described, theinvention also provides devices, systems, and methods for delivery ofsmall interfering RNA to target locations of the brain. The envisionedroute of delivery is through the use of implanted, indwelling,intraparenchymal catheters that provide a means for injecting smallvolumes of fluid containing the dsRNA of the invention directly intolocal brain tissue. Another envisioned route of delivery is through theuse of implanted, indwelling, intraventricular catheters that provide ameans for injecting small volumes of fluid containing the dsRNA of theinvention directly into cerebrospinal fluid. The proximal end of thesecatheters may be connected to an implanted, intracerebral access portsurgically affixed to the patient's cranium, or to an implanted drugpump located in the patient's torso.

Alternatively, implantable delivery devices, such as an implantable pumpmay be employed. Examples of the delivery devices within the scope ofthe invention include the Model 8506 investigational device (byMedtronic, Inc. of Minneapolis, Minn.), which can be implantedsubcutaneously on the cranium, and provides an access port through whichtherapeutic agents may be delivered to the brain. Delivery occursthrough a stereotactically implanted polyurethane catheter. Two modelsof catheters that can function with the Model 8506 access port includethe Model 8770 ventricular catheter by Medtronic, Inc., for delivery tothe intracerebral ventricles, which is disclosed in U.S. Pat. No.6,093,180, incorporated herein by reference, and the IPA1 catheter byMedtronic, Inc., for delivery to the brain tissue itself (i.e.,intraparenchymal delivery), disclosed in U.S. Ser. Nos. 09/540,444 and09/625,751, which are incorporated herein by reference. The lattercatheter has multiple outlets on its distal end to deliver thetherapeutic agent to multiple sites along the catheter path. In additionto the aforementioned device, the delivery of the small interfering RNAvectors in accordance with the invention can be accomplished with a widevariety of devices, including but not limited to U.S. Pat. Nos.5,735,814, 5,814,014, and 6,042,579, all of which are incorporatedherein by reference. Using the teachings of the invention and those ofskill in the art will recognize that these and other devices and systemsmay be suitable for delivery of small interfering RNA vectors for thetreatment of neurodegenerative diseases in accordance with theinvention.

In one such embodiment, the method further comprises the steps ofimplanting a pump outside the brain, the pump coupled to a proximal endof the catheter, and operating the pump to deliver the predetermineddosage of the at least one small interfering RNA or small interferingRNA vector through the discharge portion of the catheter. A furtherembodiment comprises the further step of periodically refreshing asupply of the at least one small interfering RNA or small interferingRNA vector to the pump outside said brain.

Thus, the invention includes the delivery of small interfering RNAvectors using an implantable pump and catheter, like that taught in U.S.Pat. Nos. 5,735,814 and 6,042,579, and further using a sensor as part ofthe infusion system to regulate the amount of small interfering RNAvectors delivered to the brain, like that taught in U.S. Pat. No.5,814,014. Other devices and systems can be used in accordance with themethod of the invention, for example, the devices and systems disclosedin U.S. Ser. No. 09/872,698 (filed Jun. 1, 2001) and Ser. No. 09/864,646(filed May 23, 2001), which are incorporated herein by reference.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulation a range of dosage for use in humans. The dosage ofcompositions of the invention lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the compound or, when appropriate, of thepolypeptide product of a target sequence (e.g., achieving a decreasedconcentration of the polypeptide) that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In addition to their administration individually or as a plurality, asdiscussed above, the dsRNAs of the invention can be administered incombination with other known agents effective in treatment of diseases.In any event, the administering physician can adjust the amount andtiming of dsRNA administration on the basis of results observed usingstandard measures of efficacy known in the art or described herein.

Methods for Treating Diseases Caused by Expression of the HD Gene

In one embodiment, the invention provides a method for treating asubject having a disease or at risk of developing a disease caused bythe expression of the HD gene, or a mutant form of the HD gene. In thisembodiment, the dsRNA acts as a therapeutic agent for controlling theexpression of the HD protein. The method comprises administering apharmaceutical composition of the invention to the patient (e.g.,human), such that expression of the HD gene is diminished at least inpart. Because of their high specificity, the dsRNAs of the inventionspecifically target mRNAs of the HD gene.

Neurodegenerative Diseases

Huntington's disease is also known as Huntington's Chorea, ChronicProgressive Chorea, and Hereditary Chorea. Huntington's disease is anautosomal dominant genetic disorder characterized by choreiformmovements and progressive intellectual deterioration, usually beginningin middle age (35 to 50 yr). The disease affects both sexes equally. Thecaudate nucleus atrophies, the small-cell population degenerates, andlevels of the neurotransmitters gamma-aminobutyric acid (GABA) andsubstance P decrease. This degeneration results in characteristic“boxcar ventricles” seen on CT scans.

The gene involved in Huntington's disease (IT-15) is located at the endof the short arm of chromosome 4. A mutation occurs in the coding regionof this gene and produces an unstable expanded trinucleotide repeat(cytosine-adenosine-guanosine), resulting in a protein with an expandedglutamate sequence. The normal and abnormal functions of this protein(termed huntingtin) are unknown. The abnormal huntingtin protein appearsto accumulate in neuronal nuclei of transgenic mice, but the causalrelationship of this accumulation to neuronal death is uncertain.

By “Huntingtin” or “HD” as used herein is meant, any Huntingtin protein,peptide, or polypeptide associated with the development or maintenanceof Huntington disease. The terms “Huntingtin” and “HD” also refer tonucleic acid sequences encoding any huntingtin protein, peptide, orpolypeptide, such as Huntingtin RNA or Huntingtin DNA (see for exampleVan Dellen et al., Jan. 24, 2004, Neurogenetics).

Symptoms and signs develop insidiously. Dementia or psychiatricdisturbances, ranging from apathy and irritability to full-blown bipolaror schizophreniform disorder, may precede the movement disorder ordevelop during its course. Anhedonia or asocial behavior may be thefirst behavioral manifestation. Motor manifestations include flickingmovements of the extremities, a lilting gait, motor impersistence(inability to sustain a motor act, such as tongue protrusion), facialgrimacing, ataxia, and dystonia.

Treatment for Huntington's disease is currently not available. Thechoreic movements and agitated behaviors may be suppressed, usually onlypartially, by antipsychotics (e.g., chlorpromazine 100 to 900 mg/day poor haloperidol 10 to 90 mg/day po) or reserpine begun with 0.1 mg/day poand increased until adverse effects of lethargy, hypotension, orparkinsonism occur.

Another embodiment of the present invention thus provides the use of ananti-Huntingtin dsRNA administered to a human, particularly the striatumof the human brain, for the treatment of Huntington's disease

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intracranial (includingintraparenchymal and intraventricular), intrathecal, epidural,intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal,airway (aerosol), nasal, rectal, vaginal and topical (including buccaland sublingual) administration. In preferred embodiments, thepharmaceutical compositions are administered by intravenous, intrathecalor intracranial infusion or injection.

Methods for Inhibiting Expression of the HD Gene

In yet another aspect, the invention provides a method for inhibitingthe expression of the HD gene in a mammal. The method comprisesadministering a composition of the invention to the mammal such thatexpression of the target HD gene is silenced. Because of their highspecificity, the dsRNAs of the invention specifically target RNAs(primary or processed) of target HD gene. Compositions and methods forinhibiting the expression of these HD genes using dsRNAs can beperformed as described elsewhere herein.

In one embodiment, the method comprises administering a compositioncomprising a dsRNA, wherein the dsRNA comprises a nucleotide sequencewhich is complementary to at least a part of an RNA transcript of the HDgene of the mammal to be treated. When the organism to be treated is amammal such as a human, the composition may be administered by any meansknown in the art including, but not limited to oral or parenteralroutes, including intracranial (including intraparenchymal andintraventricular), intrathecal, epidural, intravenous, intramuscular,intracranial, subcutaneous, transdermal, airway (aerosol), nasal,rectal, vaginal and topical (including buccal and sublingual)administration. In preferred embodiments, the compositions areadministered by intravenous, intrathecal or intracranial infusion orinjection.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

EXAMPLES

Gene Walking of the HD Gene

ClustalW multiple alignment function of BioEdit Sequence AlignmentEditor (version 7.0.4.1) was used to generate a global alignment ofhuman (NM-002111), mouse (NM_(—)010414) and rat (U18650) mRNA sequences.

Conserved regions were identified by embedded sequence analysis functionof the software. Conserved regions were defined as sequence stretcheswith a minimum length of 19 bases for all aligned sequences containingno internal gaps. Sequence positions of conserved regions were countedaccording to the human sequence.

The siRNA design web interface at Whitehead Institute for BiomedicalResearch (http://jura.wi.mit.edu/siRNAext/) (Yuan et al., Nucl. Acids.Res. 2004 32:W130-W134) was used to identify all potential siRNAstargeting the conserved regions as well as their respective off-targethits to sequences in the human, mouse and rat RefSeq database. siRNAssatisfying the cross-reactivity criteria selected out of the candidatespool and subjected to the software embedded off-target analysis. Forthis, all selected siRNAs were analyzed in 3 rounds by the NCBI blastalgorithm against the NCBI human, mouse and rat RefSeq database.

Blast results were downloaded and analyzed in order to extract theidentity of the best off-target hit for the antisense strand as well asthe positions of occurring mismatches. All siRNA candidates were rankedaccording to predicted properties. For this, different criteria wereapplied in order to identify siRNA with the following properties:targeting human, mouse and rat sequences (cross-reactivity given),absence of stretches with more than 3 Gs in a row, absence of human,mouse or rat predicted off-target hits. The siRNAs that contained theapplied criteria were selected and synthesized (Tables 1 and 2).

As has been experienced by those working in the antisense field,ribonucleic acids are often quickly degraded by a range of nucleasespresent in virtually all biological environments, e.g. endonucleases,exonucleases etc. This vulnerability may be circumvented by chemicallymodifying these oligonucleotides such that nucleases may no longerattack. Consequently, siRNAs were synthesized with 2′-O-Methylsubstitutions (Table 2) and tested for in vitro inhibitory activity onendogenous HD gene expression (HD mRNA levels).

dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology. TABLE 1 Sequences and activities of dsRNAs tested for HD geneexpression inhibiting activity Remain- ing HD SEQ SEQ SEQ gene mRNADuplex sequence of total ID Sense strand sequence ID Antisense strand ID[% of name 19 mer taraget site NO: (5′-3′) NO: Sequence (5′-3′) NO:control] AD-10894 gaaucgagaucggauguca 1 gaaucgagaucggaugucaTT 2ugacauccgaucucgauucTT 3 28 ± 3 AD-10895 aaauccugcuuuagucgag 4aaauccugcuuuagucgagTT 5 cucgacuaaagcaggauuuTT 6 45 ± 4 AD-10896agucaguccggguagaacu 7 agucaguccggguagaacuTT 8 aguucuacccggacugacuTT 9 38± 2 AD-10897 gguuuaugaacugacguua 10 gguuuaugaacugacguuaTT 11uaacgucaguucauaaaccTT 12 11 ± 2 AD-10898 guuacggguuaauuacugu 13guuacggguuaauuacuguTT 14 acaguaauuaacccguaacTT 15 28 ± 1 AD-10899ugcuuuagucgagaaccaa 16 ugcuuuagucgagaaccaaTT 17 uugguucucgacuaaagcaTT 1833 ± 3 AD-10900 ucuguaccguugaguccca 19 ucuguaccguugagucccaTT 20ugggacucaacgguacagaTT 21 35 ± 3 AD-10901 aaauuguguuagacgguac 22aaauuguguuagacgguacTT 23 guaccgucuaacacaauuuTT 24 48 ± 6 AD-10902uggccggaaacuugcuugc 25 uggccggaaacuugcuugcTT 26 gcaagcaaguuuccggccaTT 2746 ± 5 AD-10903 guucaguuacggguuaauu 28 guucaguuacggguuaauuTT 29aauuaacccguaacugaacTT 30 32 ± 3 AD-10904 gcgggcucguuccaugauc 31gcgggcucguuccaugaucTT 32 gaucauggaacgagcccgcTT 33 31 ± 1 AD-10905gacuccgagcacuuaacgu 34 gacuccgagcacuuaacguTT 35 acguuaagugcucggagucTT 3628 ± 3 AD-10906 cgcauggucgacauccuug 37 cgcauggucgacauccuugTT 38caaggaugucgaccaugcgTT 39 37 ± 2 AD-10907 aagacgagauccucgcuca 40aagacgagauccucgcucaTT 41 ugagcgaggaucucgucuuTT 42 35 ± 1 AD-10908aagucaguccggguagaac 43 aagucaguccggguagaacTT 44 guucuacccggacugacuuTT 4542 ± 4 AD-10909 aaggccuucauagcgaacc 46 aaggccuucauagcgaaccTT 47gguucgcuaugaaggccuuTT 48 65 ± 4 AD-10910 aggccuucauagcgaaccu 49aggccuucauagcgaaccuTT 50 agguucgcuaugaaggccuTT 51 23 ± 1 AD-10911acuccgagcacuuaacgug 52 acuccgagcacuuaacgugTT 53 cacguuaagugcucggaguTT 5442 ± 4 AD-10912 uaaaggccuucauagcgaa 55 uaaaggccuucauagcgaaTT 56uucgcuaugaaggccuuuaTT 57 20 ± 1 AD-10913 ucugaaucgagaucggaug 58ucugaaucgagaucggaugTT 59 cauccgaucucgauucagaTT 60 46 ± 4 AD-10914ugaaauuguguuagacggu 61 ugaaauuguguuagacgguTT 62 accgucuaacacaauuucaTT 6335 ± 1 AD-10915 uggcucgcauggucgacau 64 uggcucgcauggucgacauTT 65augucgaccaugcgagccaTT 66 42 ± 5 AD-10916 aaagucaguccggguagaa 67aaagucaguccggguagaaTT 68 uucuacccggacugacuuuTT 69 42 ± 4 AD-10917gagugcccgugucgguucu 70 gagugcccgugucgguucuTT 71 agaaccgacacgggcacucTT 7277 ± 8 AD-10918 ggagcucgggacggauagu 73 ggagcucgggacggauaguTT 74acuauccgucccgagcuccTT 75 94 ± 9 AD-10919 agaaaacaagccuugccgc 76agaaaacaagccuugccgcTT 77 gcggcaaggcuuguuuucuTT 78 43 ± 4 AD-10920auaaucacauucguuuguu 79 auaaucacauucguuuguuTT 80 aacaaacgaaugugauuauTT 8135 ± 4 AD-10921 ucugggcaucgcuauggaa 82 ucugggcaucgcuauggaaTT 83uuccauagcgaugcccagaTT 84 26 ± 6 AD-10922 ggccuucauagcgaaccug 85ggccuucauagcgaaccugTT 86 cagguucgcuaugaaggccTT 87  32 ± 12 AD-10923cuaaaugugcucuuaggcu 88 cuaaaugugcucuuaggcuTT 89 agccuaagagcacauuuagTT 9024 ± 8 AD-10924 guuuaugaacugacguuac 91 guuuaugaacugacguuacTT 92guaacgucaguucauaaacTT 93 18 ± 8 AD-10925 uuuaugaacugacguuaca 94uuuaugaacugacguuacaTT 95 uguaacgucaguucauaaaTT 96 25 ± 3 AD-10926augaacugacguuacauca 97 augaacugacguuacaucaTT 98 ugauguaacgucaguucauTT 9920 ± 3 AD-10927 ccacaauguugugaccgga 100 ccacaauguugugaccggaTT 101uccggucacaacauuguggTT 102 20 ± 3 AD-10928 cugguggccgaagccguag 103cugguggccgaagccguagTT 104 cuacggcuucggccaccagTT 105 38 ± 1 AD-10929aauuguguuagacgguacc 106 aauuguguuagacgguaccTT 107 gguaccgucuaacacaauuTT108 39 ± 6 AD-10930 uuguguuagacgguaccga 109 uuguguuagacgguaccgaTT 110ucgguaccgucuaacacaaTT 111 30 ± 4 AD-10931 aaaacaagccuugccgcau 112aaaacaagccuugccgcauTT 113 augcggcaaggcuuguuuuTT 114 32 ± 3 AD-10932aagagcuguaccguuggga 115 aagagcuguaccguugggaTT 116 ucccaacgguacagcucuuTT117 43 ± 5 AD-10933 auaccucagguccuguuac 118 auaccucagguccuguuacTT 119guaacaggaccugagguauTT 120 36 ± 4 AD-10934 uccugcuuuagucgagaac 121uccugcuuuagucgagaacTT 122 guucucgacuaaagcaggaTT 123 35 ± 7 AD-10935cauaaucacauucguuugu 124 cauaaucacauucguuuguTT 125 acaaacgaaugugauuaugTT126 28 ± 2 AD-10936 aagcgacugucucgacaga 127 aagcgacugucucgacagaTT 128ucugucgagacagucgcuuTT 129 29 ± 3 AD-10937 ccgagcacuuaacguggcu 130ccgagcacuuaacguggcuTT 131 agccacguuaagugcucggTT 132 38 ± 5 AD-10938cuggcucgcauggucgaca 133 cuggcucgcauggucgacaTT 134 ugucgaccaugcgagccagTT135 35 ± 2 AD-10939 uugucgccggguagaaaug 136 uugucgccggguagaaaugTT 137cauuucuacccggcgacaaTT 138 47 ± 8 AD-10940 ugcaagacucacuuagucc 139ugcaagacucacuuaguccTT 140 ggacuaagugagucuugcaTT 141 56 ± 9 AD-10941gaaacagugaguccggaca 142 gaaacagugaguccggacaTT 143 uguccggacucacuguuucTT144 36 ± 4 AD-10942 aaaucccaguguuggacca 145 aaaucccaguguuggaccaTT 146ugguccaacacugggauuuTT 147 37 ± 4 AD-10943 gcuagcuccaugcuuaagc 148gcuagcuccaugcuuaagcTT 149 gcuuaagcauggagcuagcTT 150 47 ± 4 AD-10944uccaugcuuaagccuaggg 151 uccaugcuuaagccuagggTT 152 cccuaggcuuaagcauggaTT153 102 ± 12 AD-10945 ccaugcuuaagccuaggga 154 ccaugcuuaagccuagggaTT 155ucccuaggcuuaagcauggTT 156 34 ± 5 AD-10946 ucaacagcuacacacgugu 157ucaacagcuacacacguguTT 158 acacguguguagcuguugaTT 159 40 ± 5 AD-10947augugugccacugcguuuu 160 augugugccacugcguuuuTT 161 aaaacgcaguggcacacauTT162 31 ± 3 AD-10948 ugugugccacugcguuuua 163 ugugugccacugcguuuuaTT 164uaaaacgcaguggcacacaTT 165 33 ± 1 AD-10949 ucaguccggguagaacuuc 166ucaguccggguagaacuucTT 167 gaaguucuacccggacugaTT 168 58 ± S AD-10950aguccggguagaacuucag 169 aguccggguagaacuucagTT 170 cugaaguucuacccggacuTT171 34 ± 3 AD-10951 gauuguugcuauggagcgg 172 gauuguugcuauggagcggTT 173ccgcuccauagcaacaaucTT 174 46 ± 7 AD-10952 acuuguuuacgaaaugucc 175acuuguuuacgaaauguccTT 176 ggacauuucguaaacaaguTT 177 46 ± 2 AD-10953cuuguuuacgaaaugucca 178 cuuguuuacgaaauguccaTT 179 uggacauuucguaaacaagTT180 30 ± 1 AD-10954 gcuuccgcacaugccgcgg 181 gcuuccgcacaugccgcggTT 182ccgcggcaugugcggaagcTT 183 45 ± 5 AD-10955 uaauuuuaacguaacucuu 184uaauuuuaacguaacucuuTT 185 aagaguuacguuaaaauuaTT 186 104 ± 6  AD-10956cuuucuaugcccguguaaa 187 cuuucuaugcccguguaaaTT 188 uuuacacgggcauagaaagTT189 59 ± 3 AD-10957 aaagggaaggacugacgag 190 aaagggaaggacugacgagTT 191cucgucaguccuucccuuuTT 192 84 ± 4 AD-10958 gcuggcucgcauggucgac 193gcuggcucgcauggucgacTT 194 gucgaccaugcgagccagcTT 195 44 ± 4 AD-10959ugacguuacaucauacaca 196 ugacguuacaucauacacaTT 197 uguguaugauguaacgucaTT198 19 ± 3 AD-10960 acgguaccgacaaccagua 199 acgguaccgacaaccaguaTT 200uacugguugucgguaccguTT 201 25 ± 3 AD-10961 gguaccgacaaccaguauu 202gguaccgacaaccaguauuTT 203 aauacugguugucgguaccTT 204 19 ± 3 AD-10962acgagugcucaauaauguu 205 acgagugcucaauaauguuTT 206 aacauuauugagcacucguTT207 19 ± 3 AD-10963 caucggagaguuucugucc 208 caucggagaguuucuguccTT 209ggacagaaacucuccgaugTT 210 38 ± 5 AD-10964 gcgaaccugaagucaagcu 211gcgaaccugaagucaagcuTT 212 agcuugacuucagguucgcTT 213 35 ± 4 AD-10965cugaaucgagaucggaugu 214 cugaaucgagaucggauguTT 215 acauccgaucucgauucagTT216 31 ± 2 AD-10966 cgguaccgacaaccaguau 217 cgguaccgacaaccaguauTT 218auacugguugucgguaccgTT 219 26 ± 2 AD-10967 acugaaccgggugaucaag 220acugaaccgggugaucaagTT 221 cuugaucacccgguucaguTT 222 43 ± 3 AD-10968ccuugccgcaucaaaggug 223 ccuugccgcaucaaaggugTT 224 caccuuugaugcggcaaggTT225 64 ± 9

Remain- ing HD SEQ SEQ SEQ gene m Duplex sequence of total ID Sensestrand sequence ID Antisense strand ID [% of name 19 mer taraget siteNO: (5′-3′) NO: Sequence (5′-3′) NO: control] AD-10969cuuuggcggauugcauucc 226 cuuuggcggauugcauuccTT 227 ggaaugcaauccgccaaagTT228 45 ± 3 AD-10970 cuguaccguugagucccaa 229 cuguaccguugagucccaaTT 230uugggacucaacgguacagTT 231 33 ± 1 AD-10971 uguaccguugagucccaag 232uguaccguugagucccaagTT 233 cuugggacucaacgguacaTT 234 36 ± 4 AD-10972agucgagaaccaaugaugg 235 agucgagaaccaaugauggTT 236 ccaucauugguucucgacuTT237 34 ± 5 AD-10973 ccgacuaccgcuggugggc 238 ccgacuaccgcuggugggcTT 239gcccaccagcgguagucggTT 240 47 ± 7 AD-10974 auaucaccggcugcugacu 241auaucaccggcugcugacuTT 242 agucagcagccggugauauTT 243 73 ± 6 AD-10975ugcauaucgcugggcucaa 244 ugcauaucgcugggcucaaTT 245 uugagcccagcgauaugcaTT246 88 ± 1 AD-10976 uuguuuacgacgugaucua 247 uuguuuacgacgugaucuaTT 248uagaucacgucguaaacaaTT 249 66 ± 5 AD-10977 guguuagacgguaccgaca 250guguuagacgguaccgacaTT 251 ugucgguaccgucuaacacTT 252 21 ± 2 AD-10978cuugaacuacaucgaucau 253 cuugaacuacaucgaucauTT 254 augaucgauguaguucaagTT255 37 ± 6 AD-10979 ggccggaaacuugcuugca 256 ggccggaaacuugcuugcaTT 257ugcaagcaaguuuccggccTT 258 32 ± 3 AD-10980 cugucucgacagauagcug 259cugucucgacagauagcugTT 260 cagcuaucugucgagacagTT 261 26 ± 8 AD-10981gcaucgcuauggaacuuuu 262 gcaucgcuauggaacuuuuTT 263 aaaaguuccauagcgaugcTT264 11 ± 2 AD-10982 acugacguuacaucauaca 265 acugacguuacaucauacaTT 266uguaugauguaacgucaguTT 267 13 ± 4 AD-10983 cugacguuacaucauacac 268cugacguuacaucauacacTT 269 guguaugauguaacgucagTT 270 31 ± 5 AD-10984ugaaucgagaucggauguc 271 ugaaucgagaucggaugucTT 272 gacauccgaucucgauucaTT273  62 ± 13 AD-10985 uagacgguaccgacaacca 274 uagacgguaccgacaaccaTT 275ugguugucgguaccgucuaTT 276 30 ± 4 AD-10986 uugccgcaucaaaggugac 277uugccgcaucaaaggugacTT 278 gucaccuuugaugcggcaaTT 279 68 ± 6 AD-10987aacuacaucgaucauggag 280 aacuacaucgaucauggagTT 281 cuccaugaucgauguaguuTT282 61 ± 5 AD-10988 uuuggcggauugcauuccu 283 uuuggcggauugcauuccuTT 284aggaaugcaauccgccaaaTT 285 48 ± 5 AD-10989 gcuuuagucgagaaccaau 286gcuuuagucgagaaccaauTT 287 auugguucucgacuaaagcTT 288 29 ± 3 AD-10990uuuagucgagaaccaauga 289 uuuagucgagaaccaaugaTT 290 ucauugguucucgacuaaaTT291 29 ± 1 AD-10991 uagucgagaaccaaugaug 292 uagucgagaaccaaugaugTT 293caucauugguucucgacuaTT 294 36 ± 3 AD-10992 aagugucuacccaguugaa 295aagugucuacccaguugaaTT 296 uucaacuggguagacacuuTT 297 31 ± 3 AD-10993ucaguuacggguuaauuac 298 ucaguuacggguuaauuacTT 299 guaauuaacccguaacugaTT300 44 ± 8 AD-10994 uuacggguuaauuacuguc 302 uuacggguuaauuacugucTT 302gacaguaauuaacccguaaTT 303  88 ± 17 AD-10995 uacggguuaauuacugucu 304uacggguuaauuacugucuTT 305 agacaguaauuaacccguaTT 306 65 ± 5 AD-10996gucucgacagauagcugac 307 gucucgacagauagcugacTT 308 gucagcuaucugucgagacTT309 32 ± 3 AD-10997 ucucgacagauagcugaca 310 ucucgacagauagcugacaTT 311ugucagcuaucugucgagaTT 312 34 ± 2 AD-10998 ugcgggcucguuccaugau 313ugcgggcucguuccaugauTT 314 aucauggaacgagcccgcaTT 315 34 ± 4 AD-10999uucagucucguugugaaaa 316 uucagucucguugugaaaaTT 317 uuuucacaacgagacugaaTT318 37 ± 2 AD-11000 ugucgccggguagaaaugc 319 ugucgccggguagaaaugcTT 320gcauuucuacccggcgacaTT 321 91 ± 2 AD-11001 ucggaguucaaccuaagcc 322ucggaguucaaccuaagccTT 323 ggcuuagguugaacuccgaTT 324 70 ± 6 AD-11002caugcuuaagccuagggau 325 caugcuuaagccuagggauTT 326 aucccuaggcuuaagcaugTT327 37 ± 6 AD-11003 ccgcugagucuggaucucc 328 ccgcugagucuggaucuccTT 329ggagauccagacucagcggTT 330  70 ± 12 AD-11004 ugucaacagcuacacacgu 331ugucaacagcuacacacguTT 332 acguguguagcuguugacaTT 333 43 ± 4 AD-11005guggccggcaacccagcug 334 guggccggcaacccagcugTT 335 cagcuggguugccggccacTT336 40 ± 3 AD-11006 gaaagggaucgcccacugc 337 gaaagggaucgcccacugcTT 338gcagugggcgaucccuuucTT 339 42 ± 2 AD-11007 aaagggaucgcccacugcg 340aaagggaucgcccacugcgTT 341 cgcagugggcgaucccuuuTT 342 43 ± 2 AD-11008cggguagaacuucagaccc 343 cggguagaacuucagacccTT 344 gggucugaaguucuacccgTT345 33 ± 3 AD-11009 gcucgaccgcagggccuuc 346 gcucgaccgcagggccuucTT 347gaaggcccugcggucgagcTT 348 49 ± 4 AD-11010 agcccauaucaccggcugc 349agcccauaucaccggcugcTT 350 gcagccggugauaugggcuTT 351 46 ± 1 AD-11011uucuaugcccguguaaagu 352 uucuaugcccguguaaaguTT 353 acuuuacacgggcauagaaTT354 100 ± 5  AD-11012 cccuuuuagucaggagagu 355 cccuuuuagucaggagaguTT 356acucuccugacuaaaagggTT 357 94 ± 8 AD-11013 gguuggcgacugucaugug 358gguuggcgacugucaugugTT 359 cacaugacagucgccaaccTT 360 156 ± 10 AD-11014acugucucgacagauagcu 361 acugucucgacagauagcuTT 362 agcuaucugucgagacaguTT363 39 ± 5 AD-11015 uugucugacaauaugugaa 364 uugucugacaauaugugaaTT 365uucacauauugucagacaaTT 366 21 ± 1 AD-11016 cugggcaucgcuauggaac 367cugggcaucgcuauggaacTT 368 guuccauagcgaugcccagTT 369 25 ± 3 AD-11017cucggaguuugcgugcugc 370 cucggaguuugcgugcugcTT 371 gcagcacgcaaacuccgagTT372 29 ± 3 AD-11018 uguuaaaggccuucauagc 373 uguuaaaggccuucauagcTT 374gcuaugaaggccuuuaacaTT 375 42 ± 3 AD-11019 uuaaaggccuucauagcga 376uuaaaggccuucauagcgaTT 377 ucgcuaugaaggccuuuaaTT 378 32 ± 4 AD-11020gccuucauagcgaaccuga 379 gccuucauagcgaaccugaTT 380 ucagguucgcuaugaaggcTT381  26 ± 10 AD-11021 aaggcagcuucggagugac 382 aaggcagcuucggagugacTT 383gucacuccgaagcugccuuTT 384 27 ± 2 AD-11022 agguuuaugaacugacguu 385agguuuaugaacugacguuTT 386 aacgucaguucauaaaccuTT 387 10 ± 2 AD-11023aacugacguuacaucauac 388 aacugacguuacaucauacTT 389 guaugauguaacgucaguuTT390 39 ± 3 AD-11024 cacaauguugugaccggag 391 cacaauguugugaccggagTT 392cuccggucacaacauugugTT 393 23 ± 4 AD-11025 caauguugugaccggagcc 394caauguugugaccggagccTT 395 ggcuccggucacaacauugTT 396 25 ± 4 AD-11026agcagcucuucagaacgcc 397 agcagcucuucagaacgccTT 398 ggcguucugaagagcugcuTT399  74 ± 11 AD-11027 guggccgaagccguagugg 400 guggccgaagccguaguggTT 401ccacuacggcuucggccacTT 402 32 ± 4 AD-11028 cguagugggaguauugugg 403cguagugggaguauuguggTT 404 ccacaauacucccacuacgTT 405 26 ± 4 AD-11029ggaguauuguggaacuuau 406 ggaguauuguggaacuuauTT 407 auaaguuccacaauacuccTT408 20 ± 2 AD-11030 aguauuguggaacuuauag 409 aguauuguggaacuuauagTT 410cuauaaguuccacaauacuTT 411 35 ± 3 AD-11031 gagaucggaugucagcagc 412gagaucggaugucagcagcTT 413 gcugcugacauccgaucucTT 414  53 ± 18 AD-11032cagcgccgucccaucugac 415 cagcgccgucccaucugacTT 416 gucagaugggacggcgcugTT417 49 ± 4 AD-11033 ccaccgaagggccugauuc 418 ccaccgaagggccugauucTT 419gaaucaggcccuucgguggTT 420 28 ± 6 AD-11034 auuguguuagacgguaccg 421auuguguuagacgguaccgTT 422 cgguaccgucuaacacaauTT 423 111 ± 12 AD-11035ccgacaaccaguauuuggg 424 ccgacaaccaguauuugggTT 425 cccaaauacugguugucggTT426 25 ± 5 AD-11036 aaacaagccuugccgcauc 427 aaacaagccuugccgcaucTT 428gaugcggcaaggcuuguuuTT 429 35 ± 4 AD-11037 gccuugccgcaucaaaggu 430gccuugccgcaucaaagguTT 431 accuuugaugcggcaaggcTT 432 36 ± 9 AD-11038aucuugaacuacaucgauc 433 aucuugaacuacaucgaucTT 434 gaucgauguaguucaagauTT435 40 ± 5 AD-11039 aucgaucauggagacccac 436 aucgaucauggagacccacTT 437gugggucuccaugaucgauTT 438 69 ± 5 AD-11040 uggagacccacagguucga 439uggagacccacagguucgaTT 440 ucgaaccugugggucuccaTT 441 39 ± 9 AD-11041ggagacccacagguucgag 442 ggagacccacagguucgagTT 443 cucgaaccugugggucuccTT444  65 ± 14 AD-11042 ccgcuuccacgugggagau 445 ccgcuuccacgugggagauTT 446aucucccacguggaagcggTT 447 63 ± 2 AD-11043 ucuuuggcggauugcauuc 448ucuuuggcggauugcauucTT 449 gaaugcaauccgccaaagaTT 450 60 ± 5 AD-11044uuggcggauugcauuccuu 451 uuggcggauugcauuccuuTT 452 aaggaaugcaauccgccaaTT453 30 ± 2 AD-11045 agcagcuacagugaguuag 454 agcagcuacagugaguuagTT 455cuaacucacuguagcugcuTT 456 64 ± 2 AD-11046 cgagugcucaauaauguug 457cgagugcucaauaauguugTT 458 caacauuauugagcacucgTT 459 18 ± 5 AD-11047aauuaggcuugucccaaag 460 aauuaggcuugucccaaagTT 461 cuuugggacaagccuaauuTT462  54 ± 14 AD-11048 uggaguuuagguuggcacu 463 uggaguuuagguuggcacuTT 464agugccaaccuaaacuccaTT 465 44 ± 5 AD-11049 cuugguucccauuggaucu 466cuugguucccauuggaucuTT 467 agauccaaugggaaccaagTT 468 32 ± 4 AD-11050uuuuggccggaaacuugcu 469 uuuuggccggaaacuugcuTT 470 agcaaguuuccggccaaaaTT471  53 ± 12 AD-11051 ugccuucucuaacaaaccc 472 ugccuucucuaacaaacccTT 473ggguuuguuagagaaggcaTT 474 57 ± 5 AD-11052 uaagucccauccgacgaaa 475uaagucccauccgacgaaaTT 476 uuucgucggaugggacuuaTT 477 43 ± 4 AD-11053ugauaccucagguccuguu 478 ugauaccucagguccuguuTT 479 aacaggaccugagguaucaTT480 26 ± 2 AD-11054 gauaccucagguccuguua 481 gauaccucagguccuguuaTT 482uaacaggaccugagguaucTT 483 30 ± 5 AD-11055 uguuacaacaaguaaaucc 484uguuacaacaaguaaauccTT 485 ggauuuacuuguuguaacaTT 486 81 ± 4 AD-11056cuaggauaccugaaauccu 487 cuaggauaccugaaauccuTT 488 aggauuucagguauccuagTT489  35 ± 13 AD-11057 cuuuagucgagaaccaaug 490 cuuuagucgagaaccaaugTT 491cauugguucucgacuaaagTT 492 33 ± 6 AD-11058 acuguuuguguucaacaau 493acuguuuguguucaacaauTT 494 auuguugaacacaaacaguTT 495 39 ± 4 AD-11059caauuguugaagacucucu 496 caauuguugaagacucucuTT 497 agagagucuucaacaauugTT498 39 ± 3 AD-11060 caagucacaaggccgagca 499 caagucacaaggccgagcaTT 500ugcucggccuugugacuugTT 501 40 ± 1 AD-11061 aagucacaaggccgagcac 502aagucacaaggccgagcacTT 503 gugcucggccuugugacuuTT 504 38 ± 5 AD-11062ggcuuguaccacuacugcu 505 ggcuuguaccacuacugcuTT 506 agcaguagugguacaagccTT507 27 ± 3 AD-11063 acgacaccucgggaugguu 508 acgacaccucgggaugguuTT 509aaccaucccgaggugucguTT 510 38 ± 4 AD-11064 caccucgggaugguuugau 511caccucgggaugguuugauTT 512 aucaaaccaucccgaggugTT 513  52 ± 11 AD-11065cucgggaugguuugauguc 514 cucgggaugguuugaugucTT 515 gacaucaaaccaucccgagTT516  49 ± 13 AD-11066 agugucacaaagaaccgug 517 agugucacaaagaaccgugTT 518cacgguucuuugugacacuTT 519  43 ± 13 AD-11067 gugucacaaagaaccgugc 520gugucacaaagaaccgugcTT 521 gcacgguucuuugugacacTT 522 30 ± 6 AD-11068aaccgugcagauaagaaug 523 aaccgugcagauaagaaugTT 524 cauucuuaucugcacgguuTT525 36 ± 7 AD-11069 accgugcagauaagaaugc 526 accgugcagauaagaaugcTT 527gcauucuuaucugcacgguTT 528 39 ± 3 AD-11070 ccgugcagauaagaaugcu 529ccgugcagauaagaaugcuTT 530 agcauucuuaucugcacggTT 531 39 ± 3 AD-11071gcagauaagaaugcuauuc 532 gcagauaagaaugcuauucTT 533 gaauagcauucuuaucugcTT534 37 ± 4 AD-11072 acauucguuuguuugaacc 535 acauucguuuguuugaaccTT 536gguucaaacaaacgaauguTT 537 62 ± 3 AD-11073 ugaaccucuuguuauaaaa 538ugaaccucuuguuauaaaaTT 539 uuuuauaacaagagguucaTT 540 21 ± 4 AD-11074uuuagauuugcuggcgcag 541 uuuagauuugcuggcgcagTT 542 cugcgccagcaaaucuaaaTT543 80 ± 5 AD-11075 ugguucaguuacggguuaa 544 ugguucaguuacggguuaaTT 545uuaacccguaacugaaccaTT 546  32 ± 13 AD-11076 gggccaguucagggaauca 547gggccaguucagggaaucaTT 548 ugauucccugaacuggcccTT 549 30 ± 7 AD-11077uggaagcgacugucucgac 550 uggaagcgacugucucgacTT 551 gucgagacagucgcuuccaTT552 41 ± 5 AD-11078 ggaagcgacugucucgaca 553 ggaagcgacugucucgacaTT 554ugucgagacagucgcuuccTT 555 30 ± 8 AD-11079 gaagcgacugucucgacag 556gaagcgacugucucgacagTT 557 cugucgagacagucgcuucTT 558 35 ± 8 AD-11080gcgacugucucgacagaua 559 gcgacugucucgacagauaTT 560 uaucugucgagacagucgcTT561 35 ± 6 AD-11081 ugucucgacagauagcuga 562 ugucucgacagauagcugaTT 563ucagcuaucugucgagacaTT 564 33 ± 4 AD-11082 cucgacagauagcugacau 565cucgacagauagcugacauTT 566 augucagcuaucugucgagTT 567 39 ± 7 AD-11083agguggaaaugagugagca 568 agguggaaaugagugagcaTT 569 ugcucacucauuuccaccuTT570 27 ± 4 AD-11084 agugagcagcaacauacuu 571 agugagcagcaacauacuuTT 572aaguauguugcugcucacuTT 573 23 ± 3 AD-11085 guuccgcagugauggcugu 574guuccgcagugauggcuguTT 575 acagccaucacugcggaacTT 576 37 ± 4 AD-11086caaccacaccgacuaccgc 577 caaccacaccgacuaccgcTT 578 gcgguagucggugugguugTT579 36 ± 5 AD-11087 aaccacaccgacuaccgcu 580 aaccacaccgacuaccgcuTT 581agcgguagucggugugguuTT 582  48 ± 10 AD-11088 accacaccgacuaccgcug 583accacaccgacuaccgcugTT 584 cagcgguagucggugugguTT 585 42 ± 3 AD-11089cccgaaaagacacagucug 586 cccgaaaagacacagucugTT 587 cagacugugucuuuucgggTT588 37 ± 2 AD-11090 uccagcacaaaguuacuua 589 uccagcacaaaguuacuuaTT 590uaaguaacuuugugcuggaTT 591 35 ± 4 AD-11091 uuggaaugugcaauagaga 592uuggaaugugcaauagagaTT 593 ucucuauugcacauuccaaTT 594 29 ± 6 AD-11092agaucugaucagccuuucc 595 agaucugaucagccuuuccTT 596 ggaaaggcugaucagaucuTT597 43 ± 3 AD-11093 caggcaauucagucucguu 598 caggcaauucagucucguuTT 599aacgagacugaauugccugTT 600 31 ± 3 AD-11094 ggcaauucagucucguugu 601ggcaauucagucucguuguTT 602 acaacgagacugaauugccTT 603 27 ± 3 AD-11095gcaauucagucucguugug 604 gcaauucagucucguugugTT 605 cacaacgagacugaauugcTT606 23 ± 3 AD-11096 aauucagucucguugugaa 607 aauucagucucguugugaaTT 608uucacaacgagacugaauuTT 609 27 ± 3 AD-11097 ucagucucguugugaaaac 610ucagucucguugugaaaacTT 611 guuuucacaacgagacugaTT 612 42 ± 8 AD-11098aaaccuuucaacuccaacc 613 aaaccuuucaacuccaaccTT 614 gguuggaguugaaagguuuTT615 60 ± 7 AD-11099 cuuuccgugugcuggcucg 616 cuuuccgugugcuggcucgTT 617cgagccagcacacggaaagTT 618 46 ± 4 AD-11100 ccgugugcuggcucgcaug 619ccgugugcuggcucgcaugTT 620 caugcgagccagcacacggTT 621 33 ± 3 AD-11101ucgacauccuugcuugucg 622 ucgacauccuugcuugucgTT 623 cgacaagcaaggaugucgaTT624 47 ± 4 AD-11102 ugcuugucgccggguagaa 625 ugcuugucgccggguagaaTT 626uucuacccggcgacaagcaTT 627 43 ± 8 AD-11103 gcuugucgccggguagaaa 628gcuugucgccggguagaaaTT 629 uuucuacccggcgacaagcTT 630 35 ± 7 AD-11104cuugucgccggguagaaau 631 cuugucgccggguagaaauTT 632 auuucuacccggcgacaagTT633 37 ± 9 AD-11105 ggcccaguugccaauggaa 634 ggcccaguugccaauggaaTT 635uuccauuggcaacugggccTT 636 39 ± 5 AD-11106 cagguuucgucucuccacc 637cagguuucgucucuccaccTT 638 gguggagagacgaaaccugTT 639 38 ± 8 AD-11107ggcacgugucacuggaaac 640 ggcacgugucacuggaaacTT 641 guuuccagugacacgugccTT642 39 ± 3 AD-11108 cuggaaacagugaguccgg 643 cuggaaacagugaguccggTT 644ccggacucacuguuuccagTT 645 51 ± 3 AD-11109 caaaucccaguguuggacc 646caaaucccaguguuggaccTT 647 gguccaacacugggauuugTT 648 53 ± 4 AD-11110acucggaguucaaccuaag 649 acucggaguucaaccuaagTT 650 cuuagguugaacuccgaguTT651 43 ± 3 AD-11111 cucggaguucaaccuaagc 652 cucggaguucaaccuaagcTT 653gcuuagguugaacuccgagTT 654 41 ± 6 AD-11112 agccuagggaugagugaaa 655agccuagggaugagugaaaTT 656 uuucacucaucccuaggcuTT 657 34 ± 5 AD-11113gucaacagcuacacacgug 658 gucaacagcuacacacgugTT 659 cacguguguagcuguugacTT660 42 ± 4 AD-11114 gauggucacccaaaccggg 661 gauggucacccaaaccgggTT 662cccgguuugggugaccaucTT 663 49 ± 3 AD-11115 ugacagaacugcgaagggu 664ugacagaacugcgaaggguTT 665 acccuucgcaguucugucaTT 666 53 ± 8 AD-11116gaagacgagauccucgcuc 667 gaagacgagauccucgcucTT 668 gagcgaggaucucgucuucTT669 43 ± 7 AD-11117 acgagauccucgcucagua 670 acgagauccucgcucaguaTT 671uacugagcgaggaucucguTT 672 40 ± 9 AD-11118 aaccugaaagggaucgccc 673aaccugaaagggaucgcccTT 674 gggcgaucccuuucagguuTT 675 81 ± 7 AD-11119gaucgcccacugcgugaac 676 gaucgcccacugcgugaacTT 677 guucacgcagugggcgaucTT678 50 ± 7 AD-11120 cacugcgugaacauucaca 679 cacugcgugaacauucacaTT 680ugugaauguucacgcagugTT 681 40 ± 13 AD-11121 agaacuauccucuggacgu 682agaacuauccucuggacguTT 683 acguccagaggauaguucuTT 684 41 ± 8 AD-11122gucaguccggguagaacuu 685 gucaguccggguagaacuuTT 686 aaguucuacccggacugacTT687  37 ± 10 AD-11123 ugaacaaagucaucggaga 688 ugaacaaagucaucggagaTT 689ucuccgaugacuuuguucaTT 690 39 ± 6 AD-11124 aagucaucggagaguuucu 691aagucaucggagaguuucuTT 692 agaaacucuccgaugacuuTT 693 40 ± 2 AD-11125gucaucggagaguuucugu 694 gucaucggagaguuucuguTT 695 acagaaacucuccgaugacTT696 37 ± 4 AD-11126 ggccaccgugguguauaag 697 ggccaccgugguguauaagTT 698cuuauacaccacgguggccTT 699 48 ± 2 AD-11127 accgugguguauaaggugu 700accgugguguauaagguguTT 701 acaccuuauacaccacgguTT 702 36 ± 2 AD-11128cugacuuguuuacgaaaug 703 cugacuuguuuacgaaaugTT 704 cauuucguaaacaagucagTT705 33 ± 7 AD-11129 uguuuacgaaauguccaca 706 uguuuacgaaauguccacaTT 707uguggacauuucguaaacaTT 708 46 ± 8 AD-11130 ccaccgagccagcuugguc 709ccaccgagccagcuuggucTT 710 gaccaagcuggcucgguggTT 711  51 ± 12 AD-11131caccgagccagcuuggucc 712 caccgagccagcuugguccTT 713 ggaccaagcuggcucggugTT714  53 ± 15 AD-11132 caggcaacgugcgugucuc 715 caggcaacgugcgugucucTT 716gagacacgcacguugccugTT 717 46 ± 6 AD-11133 aacgugcgugucucugcca 718aacgugcgugucucugccaTT 719 uggcagagacacgcacguuTT 720 59 ± 6 AD-11134uuaauuuuaacguaacucu 721 uuaauuuuaacguaacucuTT 722 agaguuacguuaaaauuaaTT723  64 ± 16 AD-11135 uuaacguaacucuuucuau 724 uuaacguaacucuuucuauTT 725auagaaagaguuacguuaaTT 726 57 ± 6 AD-11136 uaacguaacucuuucuaug 727uaacguaacucuuucuaugTT 728 cauagaaagaguuacguuaTT 729 72 ± 9 AD-11137aacguaacucuuucuaugc 730 aacguaacucuuucuaugcTT 731 gcauagaaagaguuacguuTT732 68 ± 8 AD-11138 guaacucuuucuaugcccg 733 guaacucuuucuaugcccgTT 734cgggcauagaaagaguuacTT 735  69 ± 10 AD-11139 uaugcccguguaaaguaug 736uaugcccguguaaaguaugTT 737 cauacuuuacacgggcauaTT 738 102 ± 4  AD-11140ugcccguguaaaguaugug 739 ugcccguguaaaguaugugTT 740 cacauacuuuacacgggcaTT741 104 ± 9  AD-11141 ugagcacccgcugacauuu 742 ugagcacccgcugacauuuTT 743aaaugucagcgggugcucaTT 744 110 ± 25 AD-11142 cacccgcugacauuuccgu 745cacccgcugacauuuccguTT 746 acggaaaugucagcgggugTT 747 50 ± 4 AD-11143uuuuagucaggagagugca 748 uuuuagucaggagagugcaTT 749 ugcacucuccugacuaaaaTT750  93 ± 17 AD-11144 agccaagucauuaaaaugg 751 agccaagucauuaaaauggTT 752ccauuuuaaugacuuggcuTT 753 62 ± 4 AD-11145 guuggcgacugucaugugg 754guuggcgacugucauguggTT 755 ccacaugacagucgccaacTT 756 57 ± 4 AD-11146gcccuuaagggaagcuacu 757 gcccuuaagggaagcuacuTT 758 aguagcuucccuuaagggcTT759 74 ± 5 AD-11147 gcauaucgcugggcucaac 760 gcauaucgcugggcucaacTT 761guugagcccagcgauaugcTT 762  61 ± 10 AD-11148 aauaugagcucauuaguaa 763aauaugagcucauuaguaaTT 764 uuacuaaugagcucauauuTT 765 61 ± 8 AD-11149gugcccgugucgguucuuc 766 gugcccgugucgguucuucTT 767 gaagaaccgacacgggcacTT768 66 ± 5 AD-11150 aaugaaaccaggguagaau 769 aaugaaaccaggguagaauTT 770auucuacccugguuucauuTT 771 101 ± 7  AD-11151 cacccagaauguagcaucu 772cacccagaauguagcaucuTT 773 agaugcuacauucugggugTT 774 98 ± 8 AD-11152gagcucgggacggauagua 775 gagcucgggacggauaguaTT 776 uacuauccgucccgagcucTT777 77 ± 2 AD-11153 ugacaacugaaggcaaccu 778 ugacaacugaaggcaaccuTT 779agguugccuucaguugucaTT 780 86 ± 3 AD-11154 caacguggaccugccuacg 781caacguggaccugccuacgTT 782 cguaggcagguccacguugTT 783 86 ± 4 AD-11155gacugacgagagauguaua 784 gacugacgagagauguauaTT 785 uauacaucucucgucagucTT786 72 ± 2 AD-11156 acgagagauguauauuuaa 787 acgagagauguauauuuaaTT 788uuaaauauacaucucucguTT 789 63 ± 3

TABLE 2 Sequences and activities of dsRNAs with stabilizingmodifications tested for HD gene expression inhibiting activityRemaining SEQ SEQ HD gene Sense strand sequence ID Antisense strandsequence ID mRNA [% of Duplex name (5′-3′) NO: (5′-3′) NO: controls]AL-DP-5996 cmumumumagumcmgagaacmcmaaumgTT 790 cmauugguucucgacumaaagTT791 24 ± 7 AL-DP-5997 gumcmacmaaagaacmcmgumgcmagTT 792cugcmacgguucuuugugacTT 793 21 ± 5 AL-DP-5998umcmggagumumcmaacmcmumaagcmcmTT 794 ggcuumagguugaacuccgaTT 795 36 ± 9AL-DP-5999 gaaaumcmcmumgcmumumumagumcmgaTT 796 ucgacumaaagcmaggauuucTT797 20 ± 4 AL-DP-6000 umcmcmumgcmumumumagumcmgagaacmTT 798guucucgacumaaagcmaggaTT 799 22 ± 3 AL-DP-6001umumagumcmgagaacmcmaaumgaumTT 800 aucmauugguucucgacumaaTT 801 23 ± 7AL-DP-6002 umagumcmgagaacmcmaaumgaumgTT 802 cmaucmauugguucucgacumaTT 80320 ± 7 AL-DP-6003 cmumgcmumumumagumcmgagaacmcmaTT 804ugguucucgacumaaagcmagTT 805 26 ± 4 AL-DP-6004cmgcmumgcmacmcmgacmcmaaagaaTT 806 uucuuuggucggugcmagcgTT 807 42 ± 7AL-DP-6005 umgcmumumumagumcmgagaacmcmaaTT 808 uugguucucgacumaaagcmaTT809 21 ± 8 AL-DP-6006 gaacmumacmaumcmgaumcmaumggaTT 810uccmaugaucgaugumaguucTT 811 21 ± 6 AL-DP-6007umgaacmumacmaumcmgaumcmaumggTT 812 ccmaugaucgaugumaguucmaTT 813 21 ± 3AL-DP-6008 cmaaagaacmcmgumgcmagaumaaTT 814 uumaucugcmacgguucuuugTT 81521 ± 8 AL-DP-6009 cmcmcmacmumgcmgumgaacmaumumcmTT 816ugaauguucmacgcmagugggTT 817 22 ± 4 AL-DP-6010umumumagumcmgagaacmcmaaumgaTT 818 ucmauugguucucgacumaaaTT 819 31 ± 5AL-DP-6011 umggaaumgumumcmcmggagaaumcmTT 820 gauucuccggaacmauuccmaTT 82126 ± 4 AL-DP-6012 cmggagumumcmaacmcmumaagcmcmumTT 822aggcuumagguugaacuccgTT 823 28 ± 6 AL-DP-6013umggcmaumumumgaumcmcmaumgagcmTT 824 gcucmauggaucmaaaugccmaTT 825  34± 11 AL-DP-6014 umcmumggaaumgumumcmcmggagaaTT 826uucuccggaacmauuccmagaTT 827 23 ± 7 AL-DP-6015ggcmumgcmaaaumumumacmagagcmTT 828 gcucugumaaauuugcmagccTT 829 29 ± 5AL-DP-6016 gcmgumgaacmaumumcmacmagcmcmaTT 830 uggcugugaauguucmacgcTT 83117 ± 5 AL-DP-6017 umcmcmaggumumumaumgaacmumgacmTT 832gucmaguucmaumaaaccuggaTT 833 19 ± 5 AL-DP-6018aggcmaaagumgcmumcmumumaggaTT 834 uccumaagagcmacuuugccuTT 835 22 ± 6AL-DP-6019 aacmumacmaumcmgaumcmaumggagTT 836 cuccmaugaucgaugumaguuTT 837 59 ± 10 AL-DP-6020 cmaumumggaaumumcmcmumaaaaumcmTT 838gauuuumaggaauuccmaaugTT 839 19 ± 11 AL-DP-6021aumcmcmumgcmumumumagumcmgagaaTT 840 uucucgacumaaagcmaggauTT 841 35 ± 9AL-DP-6022 acmumacmaumcmgaumcmaumggagaTT 842 ucuccmaugaucgaugumaguTT 843 35 ± 18 AL-DP-6023 aaumcmcmumgcmumumumagumcmgagaTT 844ucucgacumaaagcmaggauuTT 845 26 ± 16 AL-DP-6024umgumcmcmaggumumumaumgaacmumgTT 846 cmaguucmaumaaaccuggacmaTT 847 16 ± 5AL-DP-6025 cmumcmggagumumcmaacmcmumaagcmTT 848 gcuumagguugaacuccgagTT849 24 ± 6 AL-DP-6026 umgaaaumcmcmumgcmumumumagumcmgTT 850cgacumaaagcmaggauuucmaTT 851 21 ± 6 AL-DP-6027cmagcmumumgumcmcmaggumumumaumgTT 852 cmaumaaaccuggacmaagcugTT 853 22 ± 6AL-DP-6028 cmgumgaacmaumumcmacmagcmcmagTT 854 cuggcugugaauguucmacgTT 855 33 ± 11 AL-DP-6029 cmumggcmumcmgcmaumggumcmgacmaTT 856ugucgaccmaugcgagccmagTT 857  45 ± 15 AL-DP-6030agcmumumgumcmcmaggumumumaumgaTT 858 ucmaumaaaccuggacmaagcuTT 859  75± 15 AL-DP-6031 ggcmaaagumgcmumcmumumaggagTT 860 cuccumaagagcmacuuugccTT861  28 ± 10 AL-DP-6032 gaumcmaumumggaaumumcmcmumaaaTT 862uuumaggaauuccmaaugaucTT 863 25 ± 10 AL-DP-6033cmacmumgcmgumgaacmaumumcmacmaTT 864 ugugaauguucmacgcmagugTT 865 24 ± 3AL-DP-6034 gumcmgagaacmcmaaumgaumggcmTT 866 gccmaucmauugguucucgacTT 86720 ± 1 AL-DP-6035 cmumumgumcmcmaggumumumaumgaacmTT 868guucmaumaaaccuggacmaagTT 86 9 28 ± 9 AL-DP-6036umgumgaumggcmaumcmaumggcmcmaTT 870 uggccmaugaugccmaucmacmaTT 871  50± 14 AL-DP-6037 cmacmaaagaacmcmgumgcmagaumTT 872 aucugcmacgguucuuugugTT873 20 ± 5

siRNA Synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scaleof 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems,Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass(CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support.RNA and RNA containing 2′-O-methyl nucleotides were generated by solidphase synthesis employing the corresponding phosphoramidites and2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH,Hamburg, Germany). These building blocks were incorporated at selectedsites within the sequence of the oligoribonucleotide chain usingstandard nucleoside phosphoramidite chemistry such as described inCurrent protocols in nucleic acid chemistry, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioatelinkages were introduced by replacement of the iodine oxidizer solutionwith a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) inacetonitrile (1%). Further ancillary reagents were obtained fromMallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anionexchange HPLC were carried out according to established procedures.Yields and concentrations were determined by UV absorption of a solutionof the respective RNA at a wavelength of 260 nm using a spectralphotometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany).Double stranded RNA was generated by mixing an equimolar solution ofcomplementary strands in annealing buffer (20 mM sodium phosphate, pH6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3minutes and cooled to room temperature over a period of 3-4 hours. Theannealed RNA solution was stored at −20° C. until use.

For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referredto as -Chol or -sChol, depending on whether the link to the cholesterylgroup is effected via a phosphodiester or a phosporothioate diestergroup), an appropriately modified solid support was used for RNAsynthesis. The modified solid support was prepared as follows:

Diethyl-2-azabutane-1,4-dicarboxylate AA

A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into astirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g,0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole)was added and the mixture was stirred at room temperature untilcompletion of the reaction was ascertained by TLC. After 19 h thesolution was partitioned with dichloromethane (3×100 mL). The organiclayer was dried with anhydrous sodium sulfate, filtered and evaporated.The residue was distilled to afford AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionicacid ethyl ester AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved indichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde(3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It wasthen followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). Thesolution was brought to room temperature and stirred further for 6 h.Completion of the reaction was ascertained by TLC. The reaction mixturewas concentrated under vacuum and ethyl acetate was added to precipitatediisopropyl urea. The suspension was filtered. The filtrate was washedwith 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. Thecombined organic layer was dried over sodium sulfate and concentrated togive the crude product which was purified by column chromatography (50%EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionicacid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidinein dimethylformamide at 0° C. The solution was continued stirring for 1h. The reaction mixture was concentrated under vacuum, water was addedto the residue, and the product was extracted with ethyl acetate. Thecrude product was purified by conversion into its hydrochloride salt.

3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionicacid ethyl ester AD

The hydrochloride salt of3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. Thesuspension was cooled to 0° C. on ice. To the suspensiondiisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To theresulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) wasadded. The reaction mixture was stirred overnight. The reaction mixturewas diluted with dichloromethane and washed with 10% hydrochloric acid.The product was purified by flash chromatography (10.3 g, 92%).

1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of drytoluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) ofdiester AD was added slowly with stirring within 20 mins. Thetemperature was kept below 5° C. during the addition. The stirring wascontinued for 30 mins at 0° C. and 1 mL of glacial acetic acid wasadded, immediately followed by 4 g of NaH₂PO₄.H₂O in 40 mL of water Theresultant mixture was extracted twice with 100 mL of dichloromethaneeach and the combined organic extracts were washed twice with 10 mL ofphosphate buffer each, dried, and evaporated to dryness. The residue wasdissolved in 60 mL of toluene, cooled to 0° C. and extracted with three50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extractswere adjusted to pH 3 with phosphoric acid, and extracted with five 40mL portions of chloroform which were combined, dried and evaporated todryness. The residue was, purified by column chromatography using 25%ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AF

Methanol (2 mL) was added dropwise over a period of 1 h to a refluxingmixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride(0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued atreflux temperature for 1 h. After cooling to room temperature, 1 N HCl(12.5 mL) was added, the mixture was extracted with ethylacetate (3×40mL). The combined ethylacetate layer was dried over anhydrous sodiumsulfate and concentrated under vacuum to yield the product which waspurified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AG

Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5mL) in vacuo. Anhydrous pyridine (10 mL) and4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added withstirring. The reaction was carried out at room temperature overnight.The reaction was quenched by the addition of methanol. The reactionmixture was concentrated under vacuum and to the residue dichloromethane(50 mL) was added. The organic layer was washed with 1M aqueous sodiumbicarbonate. The organic layer was dried over anhydrous sodium sulfate,filtered and concentrated. The residual pyridine was removed byevaporating with toluene. The crude product was purified by columnchromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g,95%).

Succinic acidmono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)ester AH

Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40°C. overnight. The mixture was dissolved in anhydrous dichloroethane (3mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and thesolution was stirred at room temperature under argon atmosphere for 16h. It was then diluted with dichloromethane (40 mL) and washed with icecold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). Theorganic phase was dried over anhydrous sodium sulfate and concentratedto dryness. The residue was used as such for the next step.

Cholesterol Derivatised CPG AI

Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture ofdichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296g, 0.242 mmol) in acetonitrile (1.25 mL),2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) inacetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. Tothe resulting solution triphenylphosphine (0.064 g, 0.242 mmol) inacetonitrile (0.6 ml) was added. The reaction mixture turned brightorange in color. The solution was agitated briefly using a wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM)was added. The suspension was agitated for 2 h. The CPG was filteredthrough a sintered funnel and washed with acetonitrile, dichloromethaneand ether successively. Unreacted amino groups were masked using aceticanhydride/pyridine. The achieved loading of the CPG was measured bytaking UV measurement (37 mM/g).

The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamidegroup (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivativegroup (herein referred to as “5′-Chol-”) was performed as described inWO 2004/065601, except that, for the cholesteryl derivative, theoxidation step was performed using the Beaucage reagent in order tointroduce a phosphorothioate linkage at the 5′-end of the nucleic acidoligomer.

Nucleic acid sequences are represented below using standardnomenclature, and specifically the abbreviations of Table 3. TABLE 3Abbreviations of nucleotide monomers used in nucleic acid sequencerepresentation. It will be understood that these monomers, when presentin an oligonucleotide, are mutually linked by 5′-3′-phosphodiesterbonds. Abbreviation^(a) Nucleotide(s) A, a2′-deoxy-adenosine-5′-phosphate, adenosine-5′-phosphate C, c2′-deoxy-cytidine-5′-phosphate, cytidine-5′-phosphate G, g2′-deoxy-guanosine-5′-phosphate, guanosine-5′-phosphate T, t2′-deoxy-thymidine-5′-phosphate, thymidine-5′-phosphate U, u2′-deoxy-uridine-5′-phosphate, uridine-5′-phosphate N, n any2′-deoxy-nucleotide/nucleotide (G, A, C, or T, g, a, c or u) am2′-O-methyladenosine-5′-phosphate cm 2′-O-methylcytidine-5′-phosphate gm2′-O-methylguanosine-5′-phosphate tm 2′-O-methyl-thymidine-5′-phosphateum 2′-O-methyluridine-5′-phosphate Af2′-fluoro-2′-deoxy-adenosine-5′-phosphate Cf2′-fluoro-2′-deoxy-cytidine-5′-phosphate Gf2′-fluoro-2′-deoxy-guanosine-5′-phosphate Tf2′-fluoro-2′-deoxy-thymidine-5′-phosphate Uf2′-fluoro-2′-deoxy-uridine-5′-phosphate A, C, G, T, underlined:nucleoside-5′-phosphorothioate U, a, c, g, t, u am, cm, gm, underlined:2-O-methyl-nucleoside-5′-phosphorothioate tm, um^(a)capital letters represent 2′-deoxyribonucleotides (DNA), lower caseletters represent ribonucleotides (RNA)

Screen of HD dsRNAs Against Endogenous Human HD mRNA Expression in HeLaCells

HeLa cells were obtained from American Type Culture Collection(Rockville, Md.) and cultured in Ham's F12 (Biochrom AG, Berlin,Germany) supplemented to contain 10% fetal calf serum (FCS) (BiochromAG, Berlin, Germany), Penicillin 100 U/ml, Streptomycin 100 μg/ml(Biochrom AG, Berlin, Germany) at 37° C. in an atmosphere with 5% CO₂ ina humidified incubator (Heraeus HERAcell, Kendro Laboratory Products,Langenselbold, Germany).

For transfection with siRNA, HeLa cells were seeded at a density of2.0×10⁴ cells/well in 96-well plates and transfected directly.Transfection of siRNA (30 nM for single dose screen) was carried outwith oligofectamine (Invitrogen GmbH, Karlsruhe, Germany) as describedby the manufacturer. For dose-response curves, siRNA concentrationsranged from 30 nM to 14 pM in 3-fold dilutions.

24 hours after transfection, HeLa cells were lysed and Huntingtin mRNAlevels were quantified with the Quantigene Explore Kit (Genosprectra,Dumbarton Circle Fremont, USA) according to the protocol. HuntingtinmRNA levels were normalized to GAPDH mRNA. For each siRNA, fourindividual datapoints were collected. An siRNA duplex unrelated to theHD gene was used as a control (‘VEGF ctrl’). The activity of a givenHD-specific siRNA duplex was expressed as percent HD mRNA concentrationin treated cells relative to huntingtin mRNA concentration in cellstreated with the control siRNA duplex.

Table 1 provides the results from four independent experiments of the invitro HeLa screen where the siRNAs, the sequences of which are given inTable 1, were tested at a single dose of 30 nM. The percentage of HDmRNA remaining in treated cells compared to controls, ±standarddeviation, is indicated in the rightmost column of Table 1. FIG. 1provides a graph of the results from two independent experiments of thein vitro HeLa screen where siRNAs, the sequences of which are given inTable 2, were tested at a single dose of 30 nM. In Table 2, duplex namesare given as AL-DP-xxxx whereas the same duplex in FIG. 1 is indicatedby ‘xxxx’ only. For instance, AL-DP-5997 in Table 2 corresponds to‘5997’ in FIG. 1. Again, the percentage of HD mRNA remaining in treatedcells compared to controls, ±standard deviation, is indicated in therightmost column of Table 2. A number of siRNAs at 30 nM were effectiveat reducing HD mRNA levels by more than 70% in HeLa cells.

Table 4 provides the IC50, IC80 and maximum inhibition values from twoto five independent experiments for 25 selected siRNAs. Several siRNAs(AL-DP-5997, AL-DP-6000, AL-DP-6001, AL-DP-6014, AL-DP-6020 andAL-DP-6032, indicated by *) were particularly potent in thisexperimental paradigm, and exhibited IC50 values between 10 and 130 pM.TABLE 4 IC-₅₀ mean IC-₈₀ mean max. inhib. Duplex name [nM] ± SD [nM] ±SD mean[%] ± SD AL-DP-5996 1.6 ± 1.2 22 ± 9  79 ± 6 AL-DP-5997* 0.05 ±0.02 2 ± 1 86 ± 5 AL-DP-5999 0.3 ± 0.3 8 ± 4 82 ± 4 AL-DP-6000* 0.1 ±0.1 5 ± 3 80 ± 2 AL-DP-6001* 0.1 ± 0.1 3 ± 1 83 ± 1 AL-DP-6002 0.3 ± 0.29 ± 4 78 ± 3 AL-DP-6003 0.3 ± 0.2 3 ± 2 83 ± 3 AL-DP-6005 0.3 ± 0.3 9 ±9 77 ± 7 AL-DP-6006 0.5 ± 0.1 8 ± 5 81 ± 2 AL-DP-6007 0.2 ± 0.1 5 ± 3 77± 8 AL-DP-6008 0.16 13.56 75 AL-DP-6014* 0.1 ± 0.1 6 ± 3 81 ± 6AL-DP-6016 0.2 ± 0.3  8 ± 10 81 ± 8 AL-DP-6017 0.4 ± 0.1 5 ± 4 82 ± 2AL-DP-6018  0.2 ± 0.04 7 ± 1 81 ± 3 AL-DP-6020* 0.009 ± 0.01  1 ± 1 88 ±5 AL-DP-6024 0.3 ± 0.1 6 ± 4 88 ± 1 AL-DP-6025 0.3 ± 0.3 11 ± 8  80 ± 1AL-DP-6026 0.2 ± 0.2 5 ± 4 81 ± 4 AL-DP-6027 0.5 ± 0.1 8 ± 6 81 ± 2AL-DP-6032* 0.016 ± 0.01  3 ± 5 87 ± 7 AL-DP-6033 0.3 ± 0.2 6 ± 2 78 ± 3AL-DP-6034  0.7 ± 0.03 10 ± 3  77 ± 4 AL-DP-6035 0.8 ± 0.9 7 ± 5  80 ±11 AL-DP-6037 0.2 ± 0.1 8 ± 7 79 ± 6

Screen of Selected HD dsRNAs Against Endogenous HD mRNA Expression inNeuroscreen and U87MG Cells

Neuroscreen cells (a PC12 sub-clone) were obtained from Cellomics(Pittsburgh, Pa.) and cultured in RPMI 1640 (Biochrom AG, Berlin,Germany) supplemented to contain 5% fetal calf serum (FCS) (Biochrom AG,Berlin, Germany), 10% DHS (Biochrom AG, Berlin, Germany), Penicillin 100U/ml, Streptomycin 100 μg/ml (Biochrom AG, Berlin, Germany) and 2 mML-glutamine (Biochrom AG, Berlin, Germany) at 37° C. in an atmospherewith 5% CO₂ in a humidified incubator (Heraeus HERAcell, KendroLaboratory Products, Langenselbold, Germany).

U87MG cells were obtained from American Type Culture Collection(Rockville, Md.) and cultured in Ham's F12 (Biochrom AG, Berlin,Germany) supplemented to contain 10% fetal calf serum (FCS) (BiochromAG, Berlin, Germany), Penicillin 100 U/ml, Streptomycin 100 μg/ml(Biochrom AG, Berlin, Germany) at 37° C. in an atmosphere with 5% CO₂ ina humidified incubator (Heraeus HERAcell, Kendro Laboratory Products,Langenselbold, Germany).

Transfection of Neuroscreen and U87MG cells with six selected siRNAs(AL-DP-5997, AL-DP-6000, AL-DP-6001, AL-DP-6014, AL-DP-6020 andAL-DP-6032), and quantitation of Huntingtin and GAPDH mRNA levels withthe Quantigene Explore Kit were performed in a similar manner to thatdescribed for HeLa cells.

IC50 values are provided in Table 5. In both Neuroscreen (rat) and U87MG(human) cells, IC50s were higher than in HeLa cells, in general. Of thesix siRNAs tested, AL-DP-6014 was significantly less potent than theother five siRNAs (AL-DP-5997, AL-DP-6000, AL-DP-6001, AL-DP-6020 andAL-DP-6032) against HD mRNA in Neuroscreen cells, whereas AL-DP-6000 wassignificantly less potent than the other five siRNAs (AL-DP-5997,AL-DP-6001, AL-DP-6014, AL-DP-6020 and AL-DP-6032) against HD mRNA inU87MG cells. TABLE 5 Neuroscreen IC50 U87MG IC50 Duplex name mean [nM]+/− SD mean [nM] AL-DP-5997   6 ± 2.8 2.7 AL-DP-6000 11.7 ± 10   98AL-DP-6001 18 0.28 AL-DP-6014 264 ± 180 0.47 AL-DP-6020 1.42 ± 0.2  0.17AL-DP-6032 4.2 ± 2.2 0.49

dsRNAs Targeting HD Reduce Endogenous HD Protein in HeLa Cells

Hela cells were cultured and transfected as previously described with100 nM of the indicated siRNAs, including six siRNAs against HD(AL-DP-5997, AL-DP-6000, AL-DP-6001, AL-DP-6014, AL-DP-6020 andAL-DP-6032) and one control unrelated siRNA (‘ctrl’). 48 hourspost-transfection, the cells were harvested and lysed. Proteins in thelysates were separated on an 8% denaturing PAG. Huntingtin and P-actinwere detected by standard western blot protocols using antibodies thatbind to the proteins. For Huntingtin detection, the membrane was probedwith a mouse anti-huntingtin protein monoclonal antibody (Chemicon,U.K.) followed by a horseradish peroxidase-coupled goat anti-mousesecondary antibody (Santa Cruz Biotechnology, Calif.). β-actin wasdetected by anti-actin goat polyclonal IgG (Santa Cruz, Calif.) followedby a donkey anti-goat Ig-HRP secondary antibody (Santa Cruz, Calif.).

FIG. 2 provides the results. AL-DP-5997 (‘5997’), AL-DP-6000 (‘6000’),AL-DP-6001 (‘6001’), AL-DP-6014 (‘6014’), AL-DP-6020 (‘6020’) andAL-DP-6032 (‘6032’), all at 100 nM, decreased the level of Huntingtinprotein relative to the control protein β-actin, whereas the controlunrelated siRNA (‘ctrl’) had no effect on the level of either protein.These results demonstrate that dsRNAs targeting HD effectively reducenot only HD mRNA levels, but also HD protein levels.

Stability in Cerebrospinal Fluid (CSF) of Selected dsRNAs Targeting HD

Six selected siRNAs (AL-DP-5997, AL-DP-6000, AL-DP-6001, AL-DP-6014,AL-DP-6020 and AL-DP-6032) were tested for stability at 5 uM over 48 hat 37° C. in calf and swine CSF, as well as in PBS for comparison. Theincubations in CSF were stopped at 1, 2, 4, 8, 24 and 48 hours byproteinase digestion, whereas the incubation in PBS was stopped at 0 and48 hours. Filtered samples were injected onto the IEX-HPLC underdenaturing conditions, and percent recovery of each single strand wasdetermined by measuring the area under the corresponding peak, andexpressing this area relative to that obtained at 0 hours in PBS. FIG. 3and Table 6 provide the results. At least 90% of both sense andantisense strands of AL-DP-5997, AL-DP-6000 and AL-DP-6014 wererecovered in both calf and swine CSF (Table 6). In contrast, although92% of the antisense strand of AL-DP-6001 was recovered in calf CSF,only 73% of the antisense strand was recovered in swine CSF. ForAL-DP-6020 and AL-DP-6032, at least 19% of the antisense strand was notrecoverable in both calf and swine CSF. TABLE 6 % full length materialafter 48 hours calf swine AL-DP sense antisense sense antisense 5997 10399 95 101 6000 114 101 114 97 6001 100 92 100 73 6014 91 90 90 94 6020113 68 104 32 6032 95 21 103 81

The following cleavage sites for AL-DP-6020 and AL-DP-6032 were mappedby comparing the calculated theoretical masses of all probable fragmentsof both strands with the experimental masses found by MALDI-TOF. For theantisense strand of AL-DP-6020, the fragment5′-gauuuumaggaauuccmaau-cyclic-PO₄-3′ (SEQ ID NO: 874) corresponds to3′-(n-3) based on the calculated mass of 5973.5 Da, and experimentalmass of 5973.0 Da. For the antisense strand of AL-DP-6032, the fragment5′-uumaggaauuccmaaugaucTT-3′ (SEQ ID NO: 875) corresponds to 5′-(n-1)based on the calculated mass of 6355.0 Da, and experimental mass of6355.6 Da. Given these cleavage sites, 2 new duplexes were designed withadditional chemical stabilization that comprises one additional 2′-OMegroup (Table 7): AL-DP-7100 (parent is AL-DP-6020) and AL-DP-7101(parent is AL-DP-6032). TABLE 7 Sequences and Modifications of FurtherStabilized dsRNAs AL-DP-7100 and AL-DP-7101 SEQ SEQ Duplex Sense strandsequence ID Antisense strand ID name (5′-3′) NO: sequence (5′-3′) NO:A1-DP- cmaumumggaaumumcmcmumaaaaumcmTT 876 gauuuumaggaauuccmaaumgTT 8777100 A1-DP- gaumcmaumumggaaumumcmcmumaaaTT 878 umuumaggaauuccmaaugaucTT879 7101

Four selected dsRNAs (AL-DP-5997, AL-DP-6000, AL-DP-6001 and AL-DP-7100)were tested for long-term stability at 5 uM over 14 days at 37° C. inrat CSF, as well as in PBS for comparison. The incubations in CSF werecarried out for 0, 1, 3, 5, 7, 10, or 14 days whereas the incubation inPBS was carried out for 14 days. Samples were processed as describedabove. FIG. 4 shows the results. For AL-DP-6000, the 14 day CSFstability timepoint is not available, for technical reasons. All fourdsRNAs are highly stable for 10 to 14 days at 37° C. in rat CSF, with≦30% loss of antisense or sense strands.

Potency of Cholesterol-Conjugated dsRNAs Targeting HD Against EndogenousHuman HD mRNA Expression in HeLa Cells

Previous studies [Soutschek et al., 2004] had demonstrated a beneficialeffect of cholesterol conjugation on cellular uptake and/or efficacy ofsiRNA in vivo. We synthesized dsRNAs AL-DP-6982, AL-DP-6983 andAL-DP-7130 (Table 8) which are cholesterol-conjugated versions ofAL-DP-5997, AL-DP-6000 and AL-DP-7100, respectively, in order toevaluate their biological activities in vitro and in vivo. Hela cellswere cultured and transfected as previously described, with dsRNAsAL-DP-6982, AL-DP-6983, AL-DP-7130, AL-DP-5997, AL-DP-6000, andAL-DP-7100 at concentrations ranging from 30 nM to 14 pM. TABLE 8Sequences of Cholesterol-Conjugated dsRNAs AL-DP-6982, AL-DP-6983 andAL-DP-7130 SEQ SEQ Duplex Sense strand sequence ID Antisense strand IDname (5′-3′) NO: sequence (5′-3′) NO: AL-DP-gumcmacmaaagaacmcmgumgcmagTT-sChol 880 cugcmacgguucuuugugacTT 881 6982AL-DP- umcmcmumgcmumumumagumcmgagaacmTT-sChol 882guucucgacumaaagcmaggaTT 883 6983 AL-DP-cmaumumggaaumumcmcmumaaaaumcmTT-sChol 884 gauuuumaggaauuccmaaumgTT 8857130Note:’s' represents a phosphorothioate bound inbetween T and cholesterol,Chol represents cholesterol-conjugate

24 hours after transfection, HeLa cells were lysed and Huntingtin andGAPDH mRNA levels were quantified as described above. For each siRNA,four individual datapoints were collected. An siRNA duplex unrelated tothe HD gene was used as a control. The activity of a given siRNA duplextargeting HD was expressed as percent HD mRNA concentration in treatedcells relative to the HD mRNA concentration in cells treated with thecontrol siRNA duplex. XL-fit was used to calculate IC₅₀ values; the meanIC₅₀ values were calculated from three independent determinations, andare shown in Table 9. TABLE 9 Potency of Cholesterol-Conjugated dsRNAsAL-DP-6982, AL-DP-6983 and AL-DP-7130 Compared with Unconjugated dsRNAsAL-DP-5997, AL-DP-6000 and AL-DP-7100 against endogenous human HD mRNAexpression in HeLa cells Duplex name IC50 (mean, nM) AL-DP-5997 0.04AL-DP-6982 0.73 AL-DP-6000 0.24 AL-DP-6983 14.0 AL-DP-7100 0.03AL-DP-7130 0.38

The unconjugated dsRNAs exhibited expected (Table 4) potencies in vitroagainst HD mRNA. The cholesterol-conjugated dsRNAs retain biologicalactivity in vitro against HD mRNA, although the potencies are somewhatreduced compared to the unconjugated parent molecules.

In Vivo Down-Modulation of Endogenous HD mRNA Levels by CNSAdministration of Unconjugated or Cholesterol-Conjugated dsRNAsTargeting HD in Rats and Mice

To assess both the in vivo biological activity and distribution ofunconjugated or cholesterol-conjugated dsRNAs targeting HD, dsRNAsAL-DP-1997 and AL-DP-1998 (Table 10), based on AL-DP-5997, weresynthesized in which the two 2′-deoxy-thymidine-5′-phosphate nucleotidesat the 3′-end of the antisense strand (outside of the dsRNA's nucleotideregion that targets the HD mRNA) were replaced with5-bromo-2′-deoxyuridine. TABLE 10 Sequences of dsRNAs AL-DP-1997 andAL-DP-1998 SEQ SEQ Duplex Sense strand sequence ID Antisense strand IDname (5′-3′) NO: sequence (5′-3′) NO: AL-DP-1997gumcmacmaaagaacmcmgumgcmagTT 886 cugcmacgguucuuugugacBB 887 AL-DP-1998gumcmacmaaagaacmcmgumgcmagTT-Chol 888 cugcmacgguucuuugugacBB 889Note:’B' represents 5-bromo-2′-deoxyuridine, underline designatesnucleoside-5′-phosphorothioate, Chol represents cholesterol-conjugate

In rats, 1.3 mg AL-DP-1997 or AL-DP-1998, or phosphate-buffered saline(PBS, vehicle control) was administered by continuous intrastriatalinfusion over 7 days. Male Sprague-Dawley rats, approximately 250-300 gbody weight, received stereotaxic implantation of 30-gauge infusioncannulae (Plastics One, Roanok, Va.) such that unilateral injectionswere targeted to the center of the striatum (anteroposterior +0.7 mm,mediolateral +3.0 mm, relative to bregma; dorsoventral 5 mm, relative toskull surface). Mini-osmotic pumps (model 1007D) were primed overnightaccording to the manufacturer's specifications, implantedsubcutaneously, and connected via catheters, to deliver (4 rats pertreatment group) PBS, 1.1 mM AL-DP-1997 or 1.1 mM AL-DP-1998 at 0.5uL/hr over 7 days. At the end of the 7 day infusion period, animals weresacrificed, brains were removed, and ipsilateral striata encompassingthe infusion site were flash frozen. Tissue samples of about 5-30 mgeach were homogenized by sonication (BANDELIN electronic GmbH & Co. KG,Berlin, Germany) in Tissue and Cell Lysis solution (Epicentre, Madison,Wis.) containing 84 μg/ml Proteinase K (Epicentre, Madison, Wis.).Lysates were then stored at −80° C. For carrying out the bDNA assay,frozen lysates were thawed at room temperature, and Huntingtin and GAPDHmRNA were quantified using the Quantigene Explore Kit according to themanufacturer's instructions. For each tissue sample, the ratio ofHuntingtin/GAPDH (normalized Huntingtin mRNA level) was calculated as anaverage of four determinations. These ratios were then averaged toobtain a group (treatment) average. The unconjugated dsRNA, AL-DP-1997,reduced the normalized Huntingtin mRNA level by 33%, relative to the PBScontrol group, whereas the cholesterol-conjugated dsRNA, AL-DP-1998,reduced the normalized Huntingtin mRNA level by 26%, relative to the PBScontrol group. Both reductions were statistically significant (p<0.05,ANOVA with Tukey post-hoc analysis). These results demonstrate thatintrastriatal AL-DP-1997 and AL-DP-1998 are efficacious in vivo indown-modulating HD mRNA levels.

With an identical experimental paradigm, AL-DP-5997 and AL-DP-6000 werealso found to be effective in vivo in down-modulating HD mRNA levelsafter intrastriatal infusion with 1.3 mg over 7 days (0.5 uL/hr at 1.1mM) in rats. AL-DP-5997 and AL-DP-6000 reduced the normalized HuntingtinmRNA levels in striatal tissue by 34% and 36%, respectively, relative tothe PBS control group. In addition, AL-DP-5997 and AL-DP-6000 reducedthe normalized Huntingtin mRNA levels in cortical tissue by 22% and 26%respectively. These results demonstrate that these unconjugated siRNAs,after intrastriatal infusion, not only down-modulate HD mRNA levelswithin the striatum, but also in the cortex, another major brain regionwhere neuronal loss occurs in Huntington's disease and which is locatedfurther from the infusion site.

In mice, 75 ug AL-DP-1998, or phosphate-buffered saline (PBS, vehiclecontrol) was administered by a 20 minute intrastriatal infusion. MaleBalb/c mice, approximately 20-25 g body weight, received unilateralinjections of test article that were targeted to the striatum(anteroposterior +0.5 mm, mediolateral +2.0 mm, relative to bregma;dorsoventral 3.5 mm, relative to skull surface). Test articles (1.1 mM)were injected (4 animals per test article) at 0.25 uL/min. usingpre-filled, pump-regulated Hamilton micro-syringes connected to a 33gauge needle. Approximately 72 hours following the injection, animalswere sacrificed, brains were removed, and ipsilateral striataencompassing the infusion site were dissected and flash frozen. Asdescribed above for rat tissue samples, mouse tissue samples were lysed,and Huntingtin and GAPDH mRNA levels quantified. For each tissue sample,the ratio of Huntingtin/GAPDH (normalized Huntingtin mRNA level) wascalculated as an average of four determinations. These ratios were thenaveraged to obtain a group (treatment) average. Thecholesterol-conjugated dsRNA, AL-DP-1998, reduced the normalizedHuntingtin mRNA level by 33%, relative to the PBS control group, whichwas statistically significant (p<0.05, ANOVA with Tukey post-hocanalysis). These results further confirm that AL-DP-1998 is efficaciousin vivo in down-modulating HD mRNA levels. In addition, these resultsdemonstrate that a total intrastriatal dose of AL-DP-1998 as low as 75ug resulted in significant down-modulation of HD mRNA levels.

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting theexpression of a human HD gene in a cell, wherein said dsRNA comprises atleast two sequences that are complementary to each other and wherein asense strand comprises a first sequence and an antisense strandcomprises a second sequence comprising a region of complementarity whichis substantially complementary to at least a part of a mRNA encoding HD,and wherein said region of complementarity is less than 30 nucleotidesin length and wherein said dsRNA, upon contact with a cell expressingsaid HD, inhibits expression of said HD gene by at least 20%.
 2. ThedsRNA of claim 1, wherein said first sequence is selected from the groupconsisting of Tables 1, 2, 7, 8 or 10 and said second sequence isselected from the group consisting of Tables 1, 2, 7, 8 or
 10. 3. ThedsRNA of claim 1, wherein said dsRNA comprises at least one modifiednucleotide.
 4. The dsRNA of claim 2, wherein said dsRNA comprises atleast one modified nucleotide.
 5. The dsRNA of claim 3, wherein saidmodified nucleotide is chosen from the group of: a 2′-O-methyl modifiednucleotide, a nucleotide comprising a 5′-phosphorothioate group, and aterminal nucleotide linked to a cholesteryl derivative or dodecanoicacid bisdecylamide group.
 6. The dsRNA of claim 3, wherein said modifiednucleotide is chosen from the group of: a 2′-deoxy-2′-fluoro modifiednucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, anabasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modifiednucleotide, morpholino nucleotide, a phosphoramidate, and a non-naturalbase comprising nucleotide.
 7. The dsRNA of claim 3, wherein said firstsequence is selected from the group consisting of Table 2 and saidsecond sequence is selected from the group consisting of Table
 2. 8. ThedsRNA of claim 3, wherein said first sequence is selected from the groupconsisting of Tables 1, 2, 7, 8 or 10 and said second sequence isselected from the group consisting of Tables 1, 2, 7, 8 or
 10. 9. A cellcomprising the dsRNA of claim
 1. 10. A pharmaceutical composition forinhibiting the expression of the HD gene in an organism, comprising adsRNA and a pharmaceutically acceptable carrier, wherein the dsRNAcomprises at least two sequences that are complementary to each otherand wherein a sense strand comprises a first sequence and an antisensestrand comprises a second sequence comprising a region ofcomplementarity which is substantially complementary to at least a partof a mRNA encoding HD, and wherein said region of complementarity isless than 30 nucleotides in length and wherein said dsRNA, upon contactwith a cell expressing said HD, inhibits expression of said HD gene byat least 20%.
 11. The pharmaceutical composition of claim 10, whereinsaid first sequence of said dsRNA is selected from the group consistingof Tables 1, 2, 7, 8 or 10 and said second sequence of said dsRNA isselected from the group consisting of Tables 1, 2, 7, 8 or
 10. 12. Thepharmaceutical composition of claim 10, wherein said first sequence ofsaid dsRNA is selected from the group consisting of Table 2 and saidsecond sequence of said dsRNA is selected from the group consisting ofTable
 2. 13. A method for inhibiting the expression of the HD gene in acell, the method comprising: (a) introducing into the cell adouble-stranded ribonucleic acid (dsRNA), wherein the dsRNA comprises atleast two sequences that are complementary to each other and wherein asense strand comprises a first sequence and an antisense strandcomprises a second sequence comprising a region of complementarity whichis substantially complementary to at least a part of a mRNA encoding HD,and wherein said region of complementarity is less than 30 nucleotidesin length and wherein said dsRNA, upon contact with a cell expressingsaid HD, inhibits expression of said HD gene by at least 20%; and (b)maintaining the cell produced in step (a) for a time sufficient toobtain degradation of the mRNA transcript of the HD gene, therebyinhibiting expression of the HD gene in the cell.
 14. A method oftreating, preventing or managing Huntingtin disease comprisingadministering to a patient in need of such treatment, prevention ormanagement a therapeutically or prophylactically effective amount of adsRNA, wherein the dsRNA comprises at least two sequences that arecomplementary to each other and wherein a sense strand comprises a firstsequence and an antisense strand comprises a second sequence comprisinga region of complementarity which is substantially complementary to atleast a part of a mRNA encoding HD, and wherein said region ofcomplementarity is less than 30 nucleotides in length and wherein saiddsRNA, upon contact with a cell expressing said HD, inhibits expressionof said HD gene by at least 20%.
 15. A vector for inhibiting theexpression of the HD gene in a cell, said vector comprising a regulatorysequence operably linked to a nucleotide sequence that encodes at leastone strand of a dsRNA, wherein one of the strands of said dsRNA issubstantially complementary to at least a part of a mRNA encoding HD andwherein said dsRNA is less than 30 base pairs in length and wherein saiddsRNA, upon contact with a cell expressing said HD, inhibits theexpression of said HD gene by at least 20%.
 16. A cell comprising thevector of claim 15.